Advantages of 3D reconstruction in bryozoan ...

Advantages of 3D Reconstruction in Bryozoan Development Research: Tissue Formation in Germinating Statoblasts of Plumatella fungosa (Pallas, 1768) (Plumatellidae, Phylactolaemata) Stephan Handschuh1 Thomas Schwaha2 Norma Z. Neszi3 Manfred G. Walzl4 Emmy R. Wöss5 Abstract Freshwater bryozoans belonging to the Class Phylactolaemata are characterized by the internal formation of asexual dormant buds, so-called statoblasts. This study demonstrates the progress of germination of one type of statoblast – the floatoblast – in Plumatella fungosa. Based on semi-thin sections, two different stages are examined using Amira 3.1 software for 3D reconstruction. The shapes and positions of the lophophore, digestive tract, ganglion, funiculus, retractor muscles and primary bud are presented with regard to the floatoblast valves. Digital 3D reconstruction and additional virtual section planes enhance the interpretation of histological sections. This has clear advantages for morphological research.

Introduction The asexual production of internal dormant buds, so-called statoblasts, is unique to freshwater bryozoans of the Class Phylactolaemata. These resting stages can be classified into three different categories: piptoblasts, sessoblasts and floatoblasts (Mukai 1982). Statoblasts can endure unfavourable environmental periods and serve as a means of dispersal in space and time (Karlson 1992; Wöss 2005). A protective sclerotised capsule, consisting of Department of Theoretical Biology, University of Vienna, Austria. 2 Department of Theoretical Biology, University of Vienna, Austria. 3 Department of Marine Biology, University of Vienna, Austria. 4 Department of Theoretical Biology, University of Vienna, Austria. 5 Department of Freshwater Ecology, University of Vienna, Austria. 1

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a deuteroplasmatic (ventral) and a cystigenic (dorsal) valve, encloses the germinal mass. This germinal mass is divided into an outer layer of epithelial cells and an inner mass of yolk cells (Hyman 1959). Upon germination, the two valves separate along a suture and the zooid arises (Mukai et al. 1997). Various studies of statoblasts have addressed structure (Braem 1890; Brien 1953; Pourcher and d´Hondt 1987), development (Braem 1890; Oka 1891; Kraepelin 1892; von Buddenbrock 1910; Brien 1953) and germination (Braem 1890; 1913; Oka 1891), all well summarized by Mukai (1982) and Mukai et al. (1997). Detailed studies on germination were conducted on Cristatella mucedo, Pectinatella magnifica (Braem 1913) and Asajirella gelatinosa (Oka 1891), species having the largest floatoblasts within the Phylactolaemata. However, the process of tissue formation in germinating statoblasts has so far been documented solely by histological drawings based on paraffin sections. Braem’s (1890, 1913) drawings were particularly detailed, yet it is difficult to describe a complex three-dimensional process by section drawings alone.

Results and Discussion

The future goal of our research is to describe the complete floatoblast germination in Plumatella fungosa (Pallas, 1768) by a sequence of 3D models based on many developmental stages. Each of these models will be generated by reconstructing a series of semi-thin sections with the reconstruction-software Amira 3.1. Computer-based 3D reconstruction enables the use of powerful tools that simplify the understanding and interpretation of histological sections. 3D reconstruction with Amira has so far been applied in various fields of biological research (e.g. Brandt et al. 2005; Ruthensteiner et al. 2007; Walzl et al. 2004). This approach is ideal for understanding complex structures and the floatoblast germination process. The present study outlines the main advantages of this method by presenting 3D models of Plumatella fungosa at two different germination stages.

The Amira reconstruction software contains a number of tools which simplify the interpretation of complex threedimensional objects. Some tools are available even before materials are labelled (i.e. marking structures with an electronic brush). As soon as the images are aligned, the software automatically generates two more series of virtual sections by piling up one pixel row of every image of the stack. The resulting image stack can be viewed not only in the original section plane (xy-plane, x- and y-axis represent image length and width, Fig. 1.1), but in two other virtual planes as well (xz and yz planes, z- axis represents the direction of sectioning, Figs. 1.2 and 1.3). The micrograph resolution determines the quantity of sections in the two virtual planes. Although the quality of these virtual sections (Figs. 1.2 and 1.3) is not as good as that of the original micrographs (Fig. 1.1), viewing the image stack from various directions enhances understanding of the sectioned object. Creating proper 3D models requires a clear delimitation between tissues. In some cases the original section plane will not allow a precise delimitation, but the virtual sections in an alternate plane might. This is highly relevant in studying the germination of floatoblasts because tissues like the lophophore and vestibular wall closely adjoin, and a correct visualization of the lophophore might not be possible by labeling in the original section plane only. We also used this application to distinguish the shape of the ganglion, which is complicated because it lies close to the surrounding mesodermal cells (Figs. 1.1 and 1.2). Another application available after alignment is the orthoslice tool. This tool combines the original section plane and the two virtual planes, yielding a 3D view of the image stack (Fig. 1.4). This is helpful for understanding a three-dimensional object like a germinating floatoblast even before labeling the different tissues. To create a 3D model in Amira, it is sufficient to label the tissues in the original section plane, but, as mentioned above, correcting the labels in the two virtual planes allows a more precise delimitation of tissues and thus improves the model. Once the materials are labeled, there are various options for visualization. Every structure can be added or removed separately and can also be visualized at varying transparency levels. The resulting models can be rotated and cut in every possible direction. Therefore, snapshots and videos of 3D reconstructed objects enable novel representation of the germinating statoblasts. Amira-created 3D models also demonstrate the changes between two developmental stages in Plumatella fungosa floatoblast germination. In the first investigated germination stage (GS1, Fig. 2), the primary zooid’s body is entirely surrounded by the two valves, which are almost closed (Figs. 2.2–2.4). The peritoneum has already developed, and almost the whole coelom is filled with yolk spherules and yolk nuclei. In between the yolk, some mesodermal cells present the first indication of retractor muscle formation (Fig. 2.1). On each

Material and Methods In spring 2005, cystid tubes of dead Plumatella fungosa colonies containing floatoblasts were collected from the Laxenburg Pond in Lower Austria. In the laboratory, the cystid tubes were opened using sharp needles, and the floatoblasts were transferred into a jar filled with tap water. To keep floatoblasts permanently submerged, they were put under a nylon gauze mounted on a plastic ring which rested on the bottom of the jar. Higher temperatures in the laboratory accelerated floatoblast germination. As the statoblasts’ valves started to open, two different germination stages were anesthetized with chloralhydrate and fixed in Bouin’s solution (Böck 1989). After fixation they were dehydrated in a graded series of ethanol and infiltrated with Araldite in a vacuum oven using propyleneoxide as intermedium. Cured resin-blocks were serially semi-thin-sectioned (germination stage 1 at 1µm resulting in 350 sections, germination stage 2 at 2µm resulting in 170 sections) with either glass or diamond knives, using a Leica Jung Supercut 2065 or a Reichert Ultracut-S microtome. Staining was performed according to Richardson et al. (1960). Sections were analyzed and photographed digitally using a Nikon Eclipse E800 light microscope with a DS5M-U1 camera. Before loading the images into the Amira 3.1 3D reconstruction software (www.tgs.com), they were converted into grayscales, contrast-enhanced and reduced in size (800 x 600 pixels) using Adobe Photoshop CS2. To achieve maximal congruent matching, adjacent images were aligned with the Amira 3.1 alignment tool. After alignment, the valves and different developing tissues (body wall [i.e. ectoderm and peritoneum], digestive tract, funiculus, ganglion, lophophore, retractor muscles and the primary bud) were labeled manually and the image stack was resampled to reduce data volume. Based on the labeled structures, the surfaces of the 3D models were generated, smoothed and visualized. Snapshots of the 3D models were taken from different angles to convey spatial impressions of the 3D models (see also Ruthensteiner et al. 2007).

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Figure 1. Plumatella fungosa, germination stage 2 (GS2), virtual planes help interpret a three-dimensional object. 1: Micrograph of the original histological section (xy-plane), the lines indicate the plane of the two Amira-generated virtual sections (2 and 3). 2: Virtual section (xz-plane). 3: Virtual section (yzplane). 4: 3D view of the image stack using the orthoslice tool. b bud, ca caecum, co coelom, cy cystigenic valve, de deuteroplasmatic valve, ec ectoderm, g ganglion, in intestine, lo lophophore, oe oesophagus, pe peritoneum, re retractor muscle, ve vestibulum, vo vestibular opening, vw vestibular wall, ys yolk spherules. Scale bar = 100 µm.

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Figure 2. Micrograph of a histological section and 3D models of a germinating floatoblast of Plumatella fungosa in germination stage 1 (GS1). 1: Micrograph of a histological section. 2: 3D model, the arrows indicate view 3 and 4 (transparent: cy and bw). 3: 3D model (transparent: cy and de; not displayed: bw, fu and re). 4: 3D model (transparent: cy; not displayed: bw and re). aa anal area, bw body wall, cy cystigenic valve, de deuteroplasmatic valve, di digestive tract, ec ectoderm, fu funiculus, g ganglion, gi ganglion invagination, gr groove, lo lophophore, ma mouth area, pe peritoneum, re retractor muscle, ve vestibulum, vo vestibular opening, vw vestibular wall, yn yolk nucleus, ys yolk spherules. Scale bar = 100 µm.

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Figure 3. 3D models of a germinating floatoblast of Plumatella fungosa in germination stage 2 (GS2). 1: 3D model, the arrows indicate view 2 and 3 (transparent: cy and bw; not displayed: b). 2: 3D model (transparent: cy and de; not displayed: b, bw and re). 3: 3D model (transparent: bw; not displayed: re). a anus, b bud, bw body wall, ca caecum, cy cystigenic valve, de deuteroplasmatic valve, fu funiculus, g ganglion, in intestine, lo lophophore, m mouth, oe oesophagus, re retractor muscle, vo vestibular opening. Scale bar = 100 µm.

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side of the lophophore, two portions of the retractor muscle can be distinguished (Fig. 2.2). The funiculus attaches near the deuteroplasmatic valve (Fig. 2.4). The lophophore resembles a bulge with several small hillocks, the anlagen of the developing tentacles. A continuous groove divides the two arms of the lophophore (Figs. 2.2 and 2.3). On the bottom of the groove, close to the mouth area, the bladder-shaped ganglion invaginates (Figs. 2.2 and 2.4). The vestibular wall encloses the vestibulum (Fig. 2.1) and spans from the margin of the lophophore to the vestibular opening. The vestibular opening is situated in the small gap which separates the valves. The digestive tract is U-shaped (Fig. 2.4). The lumen of the future oesophagus and caecum is well developed, but it is absent in the intestinal area, and the anus ends blindly. In the later germination stage (GS2, Fig. 3), the posterior part of the primary zooid has protruded from the protective valves (Figs. 3.1 and 3.3). This protrusion represents a sticky pad, attaching the zooid to the substrate (Mukai et al. 1997). In the anterior part, the vestibular opening is situated at the gap between the two valves, as in GS1 (Figs. 1.1 and 3.3). The remaining yolk spherules are aggregated around the digestive tract, muscles and body wall (Fig. 1.1). The retractor muscle cells have differentiated into thin muscle fibres (Fig. 3.1). The funiculus is shorter than in GS1, but, as in GS1, attaches near the deuteroplasmatic valve (Fig. 3.3). The shortening of the funiculus might reflect the growth of the digestive tract. The lophophore’s shape has changed distinctly. Instead of the hillocks, 16 tentacles can be distinguished (Fig. 3.2). The coelom has extended into the tentacles; the entrances of the coelom are shown in Figures 3.1 and 3.3. The ganglion’s shape has remained unchanged (Fig. 3.3); its lumen is still in open contact to the mouth area. The digestive tract is partitioned into oesophagus, caecum and intestine with a continuous lumen. The caecum has the shape of a bulging sack (Figs. 3.1 and 3.3). In the oral region of the cystigenic valve, the first bud arises (Fig. 3.3). In summary, our results on Plumatella fungosa agree with those of Braem´s (1890, 1913) investigations on C. mucedo and P. magnifica. The 3D models definitely recreate the shape of the primary zooid’s body. Until the point of GS2, the polypid has never been evaginated, therefore the zooid is still nourished from the remaining yolk spherules. The 16 tentacles in GS2 are short and sturdy. In comparison, fully-grown zooids of Plumatella fungosa possess 40 to 58 tentacles which are much longer and thinner (Wöss 1989). In both germination stages the mouth and oesophagus are in close proximity to the cystigenic valve; the anus and intestine to the deuteroplasmatic valve (Figs. 2.4 and 3.3). This relationship of digestive

tract and valves at the time of valve separation is apparently a constant pattern in floatoblast germination, as also described by Braem (1890, 1913), Oka (1891) and Mukai et al. (1997). Our future investigations on statoblast germination will focus on the relationship between the floatoblast axes and the germination disc. The latter represents the first morphological sign of germination. Previous authors (Braem 1913; Mukai 1982; Mukai et al. 1997) reported no relationship between the axes of the floatoblast and the germination disc, possibly because the species investigated have round or almost round floatoblasts (Braem 1913). Because of the constancy of the axis relationship between polypid and valves in our germination stages (Figs. 2.3 and 3.2), a constant axis relationship at the beginning of germination seems possible. Given the oval shape of the Plumatella floatoblast valves, further investigations on P. fungosa may yield a clearer picture. Our goal is to provide a complete description of the germination process using the semi-thin sectioning technique and combining classical analysis with reconstruction methods. We will focus on lophophore and tentacle development as well as on tissue topology with regard to floatoblast and body axes.

Conclusions Computer-based 3D reconstruction contains a number of tools that help interpret three-dimensional objects. One series of sections can hardly enable complete comprehension of a complex object. Additional virtual sections offer a crucial advantage in various fields of research for two major reasons. First, they allow investigating rare or unique objects, such as old serial sections or objects now unavailable for research due to extinction or destruction of the habitats. Second, they enable the sectioning of objects with no obvious characteristics for determining the desired section plane. We therefore recommend the 3D reconstruction method for research dealing with small, complex, rare or unique objects. 3D models provide insight into tricky problems and allow impressive visualizations.

Acknowledgments This work was initiated in a histology class at the University of Vienna organized by H. Hilgers. Especially, we would like to dedicate this paper to M. Lichtnegger, who was our colleague in this project and who died in an accident on October 10, 2007. We also thank L. Rudoll for technical assistance.

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