Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens

Abstract

Background: Trichoplax adhaerens is the best-known member of the phylum Placozoa, one of the earliest-diverging metazoan phyla. It is a small disk-shaped animal that glides on surfaces in warm oceans to feed on algae. Prior anatomical studies of Trichoplax revealed that it has a simple three-layered organization with four somatic cell types.

Results: We reinvestigate the cellular organization of Trichoplax using advanced freezing and microscopy techniques to identify localize and count cells. Six somatic cell types are deployed in stereotyped positions. A thick ventral plate, comprising the majority of the cells, includes ciliated epithelial cells, newly identified lipophil cells packed with large lipid granules, and gland cells. Lipophils project deep into the interior, where they alternate with regularly spaced fiber cells whose branches contact all other cell types, including cells of the dorsal and ventral epithelium. Crystal cells, each containing a birefringent crystal, are arrayed around the rim. Gland cells express several proteins typical of neurosecretory cells, and a subset of them, around the rim, also expresses an FMRFamide-like neuropeptide.

Conclusions: Structural analysis of Trichoplax with significantly improved techniques provides an advance in understanding its cell types and their distributions. We find two previously undetected cell types, lipohil and crystal cells, and an organized body plan in which different cell types are arranged in distinct patterns. The composition of gland cells suggests that they are neurosecretory cells and could control locomotor and feeding behavior.

Figure 1. Trichoplax overview

Montages of TEM micrographs show full thickness of a Trichoplax at its edge (A) and a central region 15-µm distant (B). Ventral side faces the substrate (bottom). Five of the six principle cell types are visible. A thin layer of dorsal epithelial cells (D) transition at the edge to the pseudo-columnar ventral epithelium consisting of ventral epithelial cells (V), lipophil cells (L), and gland cells (G), some cut along their long axes, others obliquely. In the interior, fiber cells with large cell bodies (F) mingle with the cell bodies of lipophil cells. Ventral epithelial cells bear typical cilium (arrow) and microvilli (arrowhead), and contain small dense granules near the ventral membrane and a large inclusion deep inside (asterisks). Inset: Cross section through a ventral cilium and surrounding microvilli. Scale bars represent 2 µm (A,B), 200 nm (inset).

Figure 2. Lipophil Cells

(A, B) TEM and SEM images show lipophil cells (L) interspersed with epithelial cells (V) in the ventral epithelium. (A) Lipophils are filled with numerous spherical granules (S) and consistently bear a large clear spherical granule at the ventral surface. (B) Knobby contours of lipophils (L) reflect their granular content, in contrast to the smooth surfaces of ventral epithelial cells (V). (C) Ventral surface of living Trichoplax stained with fluorescent dyes. The spherical inclusions in lipophil cells take up acidophilic Lysotracker Red (left, red; FM143 membrane dye, green) and lipophilic Nile Red (right) (D, E) Single optical sections showing immunofluorescence for MAGUKs (red) and TaCDH (green). (D) Anti-MAGUK staining near the ventral surface outlines ventral epithelial cells (small profiles, nuclei in blue) and lipophils (large profiles indicated by arrows). (E) Deep in the interior, lipophil cell bodies (nuclei outlined by anti-MAGUK staining) mingle with fiber cells outlined by anti-TaCDH. Scale bars represent 2 µm (A, B), 20 µm (C), 10 µm (D–E).

Figure 3. Gland cells

(A, B) Projection of optical sections of the ventral epithelium. An antibody raised against syntaxin (A, B; Sx) strongly labels cells deployed near the edge (bottom) and, less intensely, scattered cells further from the edge. Their typical hourglass shape is revealed at the margin where they are tilted, while they appear circular when they are perpendicular to the imaging plane. (B) Cells of similar shape and appearance also immunolabel for SNAP-25 (25), synapsin (Sy), and FMRF-amide (Rf) (green; nuclei, blue; tubulin, red). (C) Electron micrograph of a ciliated gland cell reveals a population of membrane-enclosed granules (g) comparable in size with the stained granules in (B). Dense profile at right belongs to an adjacent lipophil cell. Arrowhead indicates contact between granule and surface. Scale bars represent 20 µm (A), 10 µm (B), 1 µm (C).

Figure 4. Fiber cells

(A) SEM image of interior surface exposed by removing the dorsal epithelium. Regularly spaced fiber cells (F) extend multiple tapering processes around the cell bodies of underlying ventral epithelial cells (v). Lipophil (L) is identified by its content of granules. Inset. TEM shows dense septum bisecting the cytoplasm of a process resembling that of a fiber cell. The membrane is continuous across the septum. (B) TEM micrographs show characteristic inclusions of fiber cell bodies: clusters of mitochondria (m) interspersed with smaller inclusions; a rod-shaped structure within RER that may be a bacterium (arrow); and large electron dense inclusion (upper inset). Lower inset: Immunofluorescence staining for TaCDH (green) and tubulin (red) in an optical section near edge (left). TaCDH stain outlines fibers cells, identified by their branching patterns and large inclusions (dim red, arrowhead). Red channel was digitally enhanced (gamma 0.6) to accentuate the autofluorescence of the inclusions. Tubulin antibody stains microtubules in cells and cilia around the edge. Yellow represents overlapping signals of microtubules and exterior surfaces of epithelial cells. Nuclei blue. Scale bars represent 5 µm (A), 0.5 µm (A, inset), 2 µm (B), 10 µm (B, inset).

FIGURE 5. Dorsal epithelial cells

(A) The dorsal surface is made of a single layer of thin cells containing dark granules (black arrowheads) and joined by apical junctions (white arrowhead). Small cell bodies of dorsal epithelial cells (D) are readily distinguished from a larger fiber cell (F) containing a rod-shaped inclusion and a lipophil (L) with numerous clear inclusions. Fiber cell processes contact dorsal cells (arrows). (B) En face views of dorsal epithelium after staining with rhodamine-phalloidin (left) or antibody against MAGUKs (right, red) illustrate the variable shapes of the cells. Nuclei blue. (C) SEM of dorsal epithelium broken open transversely to expose the columnar cell bodies of dorsal epithelial cells. A fiber cell (F) lies below the dorsal cells. (D) TEM micrograph shows a dorsal cell body giving rise to a flat expansion that connects to similar expansions of the adjoining cells, thus forming the thin dorsal surface of the Trichoplax. Scale bars represent 5 µm (A, C), 20 µm (B), 1 µm (D).

Figure 6. Crystal cells

TEM micrograph shows crystal cell located near the dorsal surface (D, right) with flattened nucleus (arrow) and a central inclusion flanked by two mitochondria. Crystal cell inclusions do not survive sectioning for EM, leaving a clear, rhomboid hole in the section. Inset. DIC image of a crystal (bright rhomboid inclusion) in a cell in a living Trichoplax; nuclear stain (cyan, arrow) shows flattened nucleus. Scale bars represent 2 µm.

Figure 7

Drawing summarizing Trichoplax cell types and body plan. Facing the substrate (below) is a thick ventral plate composed of: ventral epithelial cells (VEC; light yellow) each bearing a cilium and multiple microvilli; lipophil cells (brick) that contain large lipophilic inclusions, including a very large spherical inclusion near the ventral surface (lavender); and gland cells (pale green), distinguished by their contents of secretory granules and prevalence near the margin. Dorsal epithelial cells (DEC; tan) form a roof across the top from which are suspended their cell bodies surrounded by a fluid-filled space. In between the dorsal epithelium and ventral plate are fiber cells with branching processes that contact each of the other cell types. A crystal cell (pale blue) containing a birefringent crystal lies under the dorsal epithelium near the rim.