When an epithelium ceases to exist – an ultrastructural study on the fate of the embryonic coelom in Epiperipatus biolleyi (Onychophora, Peripatidae)

Introduction

Traditionally, Arthropoda and Annelida are considered to be sister taxa comprising the Articulata (e.g. Cuvier 1817; Dohle 1979; Ax 2000; Nielsen 2001; Scholtz 2002). The presence of paired coelomic cavities per segment is one of the major synapomorphies of the Articulata. According to the Articulata hypothesis, coelomic cavities have been modified in the stem lineage of the Arthropoda by the fusion of secondary (coelom) and primary body cavities (blood vascular system). In order to take this process into account, the term ‘mixocoel’ is sometimes used (Daiber 1913; Weygoldt 1986; Seifert 1995; Nielsen 2001; Scholtz 2002). Serially arranged embryonic coelomic cavities have been described in representatives of every higher euarthropod taxon (see Anderson 1973; Gilbert 1997; Schwalm 1997). Transitory coelomic cavities also occur during embryogenesis in the Onychophora (Kennel 1888; Sedgwick 1888; Evans 1901; Glen 1918; Pflugfelder 1948; Anderson 1973; Walker 1995; Bartolomaeus and Ruhberg 1999). However, a fusion between embryonic coelomic cavities and the primary body cavity was never mentioned by the authors who worked on the differentiation of the mesoderm in the Onychophora (see Kennel 1885, 1888; Sedgwick 1885, 1886, 1887, 1888; Sheldon 1887, 1888; Sclater 1888; Willey 1898; Evans 1901; Pflugfelder 1948, 1962; Manton 1949; Pflugfelder 1980; Bartolomaeus and Ruhberg 1999). Therefore, the existence of such a fusion in onychophorans has been denied or seriously doubted (Kennel 1888; Sedgwick 1888; Pflugfelder 1948, 1962, 1980; Bartolomaeus and Ruhberg 1999). Consequently, it remains unclear, whether a ‘mixocoel’ is a characteristic of all arthropods, or represents an apomorphy of the Euarthropoda only.

In order to clarify this subject, the fate of embryonic coelomic cavities has been studied in Epiperipatus biolleyi, an onychophoran species of the Peripatidae from the neotropics. This is the first time that ultrastructural data are provided on late differentiation of embryonic coelomic linings in an arthropod. Special reference is given to early nephridiogenesis. This study is part of a larger project to unravel the organogenesis in onychophorans.

Materials and Methods

Specimens of Epiperipatus biolleyi (bouvier, 1902) were collected in Las Nubes-Cascajal area, de Coronado, near San José, Costa Rica (83°57′37″ W, 10°00′18″ N, 1750–1800 m altitude), in February 1997 and maintained in captivity by H. Ruhberg. A gravid female was dissected in March 1997 at room temperature within a fixative consisting of 2.5% glutaraldehyde buffered in 0.1 m sodium cacodylate (pH 7.0). Ruthenium red was added to the fixative before fixation in order to stain components of the extracellular matrix. The embryos were removed from the uterus and kept in the same fixative for 1 h, then washed several times in 0.1 m sodium cacodylate buffer and kept therein for 1 day at 4 °C. The embryos were then postfixed in 0.2% osmium tetroxide buffered in 0.1 m sodium cacodylate, dehydrated in an acetone series, and embedded in araldite. Several embryos or certain parts of them were cut into series of silver interference-coloured sections (65–75 nm thick) with a diamond knife in a Reichert Ultracut ultramicrotome. The sections were mounted on formvar-covered single-slot copper grids and automatically stained with uranyl acetate and lead citrate in a Leica EM ultrostainer. The sections were examined with a Philips CM 100 transmission electron microscope. One significant embryo (‘coiled stage’) was selected for the present study. The diagrammatic illustration, montage of electron micrographs, and final plates were produced using the Adobe Photoshop 7.0 and Adobe Illustrator 10 software.

Characterization of Body Cavities. – Histologically, two types of body cavities can be distinguished. The coelomic (or secondary) body cavities are generally recognized by their epithelial linings. The coelomic lining cells are interconnected by apical junctions and rest on an extracellular matrix (ECM, basal lamina, lamina densa). The apico-basal polarity of coelomic lining cells might also be indicated by (rudimentary) ciliary structures situated at the apical cellular surfaces. In contrast to secondary body cavities, the lumen of a primary body cavity is never lined with epithelial cells but is merely bordered by ECM (see Rieger 1986; Ruppert 1991; Bartolomaeus 1993; Ax 1996; Bartolomaeus and Ruhberg 1999). This characterization is applied to ultrastructural data presented herein.

Results

A coiled stage-embryo is investigated in the present study (Fig. 1A). Apart from one pair of presumptive antennae no further body appendages are seen. The embryo bears 32 external swellings which decrease in size towards the posterior end. Inside the embryo, pairs of serially arranged coelomic cavities are found in front of a posterior proliferating region. They flank vacuolated walls of the presumptive gut (Fig. 1E–G). The coelomic cavities coincide in position with the external segmental swellings. Their linings are composed of polarized cells which are connected by apical junctions and rest on a basal lamina. Rudimentary cilia barely protrude from the surface of the coelomic lining cells into the coelomic cavities (Fig. 1B).

Except for the lumen of the presumptive gut and the coelomic cavities, all other hollow spaces within the embryo are lined with lamina densa (1-3). Between different cellular layers within the embryo, these hollow spaces are often reduced to narrow spaces. They are, thus, situated between the basal laminae of the corresponding epithelia (Fig. 1C). Since all these spaces are confluent with each other, and since they are lined by electron-dense matrix, they are regarded as constituents of the primary body cavity. Every cell facing the primary body cavity is lined with lamina densa. The only exception is represented by amoeboid cells. These solitary cells are found in different regions of the embryonic body (1, 2). Amoeboid cells are recognized by pseudopodia, by which they can be attached to the lamina densa of other cells (Fig. 1D).

Within the embryo, an anterior-to-posterior developmental gradient is evident with respect to the differentiation of the mesoderm (Fig. 1E–H). The posterior segments of the embryo are the least differentiated. The coelomic linings are composed of simple monolayered epithelia which represent the only mesodermal structures (Fig. 1E). The coelomic lining cells lack myofilaments and the cellular volume is largely occupied by the nucleus (Fig. 1B). Mitotic stages rarely occur within the coelomic linings. Apart from apical junctions, no other mechanical cell-cell or cell-matrix contacts are seen among coelomic lining cells. The basal lamina of the coelomic linings is thinner than that of the ectoderm (Fig. 1C). The coelomic cavities of the posterior segments appear crescent-shaped in cross-sections. Accordingly, two contiguous portions of mesoderm are distinguished: (1) the splanchnic wall which is adjacent to the endoderm, and (2) the somatic wall which lies beneath the ectoderm. The splanchnic wall is thinner than the somatic wall (Fig. 1E).

The structure of the mesoderm is more developed in the anterior segments of the embryo. The somatic wall becomes thicker, and a lateral protrusion of the coelomic cavity is formed (Fig. 1F). The ventromedian part of the somatic wall begins to migrate inwards. Here, the coelomic lining loses its contact to the ectoderm. At the same time, the dorsal and ventral walls of the presumptive gut (endoderm) begin to lose their contact with the dorsal and ventral ectoderm. Two hollow spaces are thus formed, the dorsal and the ventral sinuses, which enlarge further and extend anteriorly (Fig. 2A). The dorsal and ventral sinuses constitute the largest spaces of the primary body cavity within the embryo.

Along with the enlargement of the neuroectoderm, several folds are formed in the ventromedian part of the mesoderm which migrates further inwards (Fig. 1G). The cells of the somatic part still undergo mitoses indicating a further increase in thickness. Within the thickened, ventrolateral area of the mesoderm, a narrow canal is formed. The lumen of this canal bears numerous microvilli and cilia. Within the canal, ciliogenesis is indicated by several vestigial cilia which are still enclosed by vesicles. The shortest of the cilia project into a small vesicle which lies within the cytoplasm of canal cells. The longer the cilium the closer it is to the cellular surface (Fig. 2D). Several cilia protrude from cellular surfaces into the lumen of the canal. Only one cilium per cell is present. The lumen of the canal is continuous with the remaining coelomic cavity distally. Proximally, the canal ends blindly, its cells being overlain by ectoderm. In contrast to the somatic wall, the splanchnic coelomic lining is still a monolayer of cells (Fig. 1G). In all posterior and most of the mid-body segments (from the 8th segment backwards), the splanchnic and somatic coelomic linings are continuous.

The continuity between the splanchnic and somatic linings is lost, however, further anteriorly (within the segments 1–7). While the dorsolateral portions of the coelomic wall are still continuous, the ventral parts have become separated (Fig. 1H). The splanchnic wall extends to the ventral sinus. That part of the somatic wall which is adjacent to the ciliated canal disintegrates into several folds (1, 2). Owing to the separation of the splanchnic and somatic walls, the coelomic cavity becomes confluent with spaces of the primary body cavity. This fusion is accompanied by changes at the cellular level. The dorsal and lateral portions of the coelomic wall (including the ciliated canal) are still composed of epithelia which bear polarized cells connected by apical junctions (Fig. 2A–D). In contrast, the remnants of the ventral coelomic wall have lost their epithelial character. Their cells are unpolarized, since they lack apical junctions and are, instead, covered with lamina densa (2, 3). Accordingly, the remnants of the coelomic cavities are surrounded by epithelial and nonepithelial cells at the same time, since apical junctions and lamina densa are met within the same hollow space (2, 3).

Discussion