Hidden cell diversity in Placozoa: ultrastructural insights from Hoilungia hongkongensis

Abstract

From a morphological point of view, placozoans are among the most simple free-living animals. This enigmatic phylum is critical for our understanding of the evolution of animals and their cell types. Their millimeter-sized, disc-like bodies consist of only three cell layers that are shaped by roughly seven major cell types. Placozoans lack muscle cells and neurons but are able to move using their ciliated lower surface and take up food in a highly coordinated manner. Intriguingly, the genome of Trichoplax adhaerens, the founding member of the enigmatic phylum, has disclosed a surprising level of genetic complexity. Moreover, recent molecular and functional investigations have uncovered a much larger, so-far hidden cell-type diversity. Here, we have extended the microanatomical characterization of a recently described placozoan species-Hoilungia hongkongensis. In H. hongkongensis, we recognized the established canonical three-layered placozoan body plan but also came across several morphologically distinct and potentially novel cell types, among them novel gland cells and "shiny spheres"-bearing cells at the upper epithelium. Thus, the diversity of cell types in placozoans is indeed higher than anticipated.

Keywords: Cell types; Functional anatomy; Hoilungia hongkongensis; Morphology; Physiology; Placozoa; Signaling; Trichoplax adhaerens.

Fig. 1

Morphology of H. hongkongensis (H13), viewed from the upper (a) and lower (b) sides. Note that the animals were fixed, while they were not adhering to a substrate, thereby showing great variability in body shapes, as they can form invaginations, ripples, curls, and can variably stretch and contract. c Ciliated epithelia in H. hongkongensis (H13). The lower surface (le) is more densely ciliated than the upper surface (ue). Scale bar 50 μm in a, b; 8 μm in c

Fig. 2

Gross anatomy of H. hongkongensis (H13). (a) A SEM micrograph from the animal’s inside reveals, from top to bottom, the flat cells of the upper epithelium, the bulky cells of the middle layer, and the numerous, mostly elongated cells of the lower epithelium. Note that micro-cavities were often observed between groups of densely packed cells of the middle and lower layers. (b) At the TEM, a full cross-section micrograph of a representative animal (approximately 250 μm wide and 30 μm thick) shows the three-layer bodyplan and upper and lower epithelia merging at the edges. Framed regions of the upper and lower epithelia are shown at higher magnification in (b1, b2) and readily reflect cell diversity. In b1, the shiny sphere-containing cell shows an elongated process toward the middle layer and protrudes between two canonical flat epithelial cells. In b2, the vast majority of columnar cells are ciliated. In contrast, lipophil cells are not ciliated, and their outermost granule (“matte-finished sphere”) abbuting the surface. As illustrated in (c) at the level of the lower epithelium, all cells are joined by adherens junctions, and most bear a cilium and microvilli-like structure (see text for details). Note the ciliary root and the ciliary pit, which can be more or less deep in different cell types, as well as some unidentified glycocalyx-like material outside the membrane. FC fiber cell, GC gland cell, LC lipophil cell, LEC lower epithelial cell, UEC upper epithelial cell, SS shiny sphere. Scale bar 6 μm in a; 18 μm in b; 5 μm in b1, b2; 1 μm in c

Fig. 3

Cell diversity in the lower epithelium of H. hongkongensis (H13). (a) Classical lower epithelial cells are columnar and polarized. They often bear clusters of greyish vesicles, possibly reflecting pinocytic events (arrows). (b) Small polarized, ciliated cells present clusters of small dark vesicles near the lower surface of the animal; they likely correspond to an undescribed population of secretory cells. Cells with likely different content (c) or with a dark appearance (d, asterisk) are regularly spotted among the cells of the lower epithelium; they may not reach the lower surface of the animal. (e, e’) In some areas of the epithelium, among lower epithelial cells and lipophil cells, a population of polarized cells contain clusters of very clear vesicles close to the Golgi apparatus or reaching the surface; they may correspond to mucus-secreting cells. (f, f’) Typical gland cells—ciliated, polarized secretory-like cells with large dense vesicles—are present at the inner rim (i.e. at the periphery but not directly at the edge) of the lower epithelium. GC gland cell, LEC lower epithelial cell, Scale bar 3 μm

Fig. 4

Ultrastructure of lipophil cells in H. hongkongensis (H13). a At the SEM, lipophil cells are at times seen bulging from the lower epithelium into the fiber cell layer, often in close vicinity to fiber cells. Their shapes are usually elongated (in the lower epithelium, where they are interspersed between lower epithelial cells (c) or more complex depending on their cellular environment (b) and position in the animal. In all cases, they exhibit large, heterogeneous vacuoles— rather clear for those close to the trans-Golgi network, otherwise with various electron densities (i.e., affinity to osmium) or structural complexity, as some of the vacuoles contain membranous material (#). Their most basal vacuole (matte-finished sphere—asterisk), abutting the lower surface, is often electron-dense but sometimes also clear. Scale bar 2 μm in a; 1.5 μm in b, c

Fig. 5

Ultrastructure of cells in the middle layer of H. hongkongensis (H13). a Each fiber cell (FC) has a bulky soma and numerous thin processes (arrows); complex, heterogenous vacuoles in varying numbers; a single large mitochondrial complex (mc); and bacteria inside the endoplasmic reticulum (asterisks). b On this SEM micrograph, a group of four fiber cells (FC) lie on top of cells of the lower epithelium; fiber cells are interconnected, and their processes reach deep between epithelial cells. c Unknown small cell (#) with processes (arrowheads) were observed in the close vicinity of fiber cells (FC). Scale bar 1.5 μm in a; 5 μm in b; 1.5 μm in c

Fig. 6

Cell diversity in the upper epithelium of H. hongkongensis (H13). a At the SEM, contours of cells composing the upper epithelium can easily be made out. Most of the cells are ciliated. A globular protrusion-like structure can be seen (arrow), which probably corresponds to a shiny sphere abutting the epithelial surface. b, c Cells from the upper epithelium are essentially bound to other cells at their basal pole (toward the upper surface) by adherens junctions, and the main cell body (including the nucleus) is sunk toward the inside of the animal. They may receive local and maybe transient contact from other cells, such as fiber cells. Canonical upper epithelial cells (“1”) are elongated T-shaped ciliated epithelial cells with pigment granules. They have a large flattened surface area, are not ramified, and their nucleus occupies most of the cytoplasm. Towards the upper surface, they contain electron-dense inclusions. c A variety of other cell types can be identified in the upper epithelium and directly underneath. Some cells (“2”), “sphere cells,” have a large inclusion that might correspond to a shiny sphere; they have no pigment granules, a small nucleus, and a few processes; some cells (“3”) contain small vesicles, reach the surface via a narrow neck, and have no cilium (see Fig. S3 for more details); right underneath the cells forming the surface, populations of small putative secretory cells with numerous small dense vesicles (“4”) or small clear vesicles (“5”) are observed; small rounded cells are seen in pairs (“6”) that may have recently duplicated; other cells, reminiscent of macrophages with an atypical nucleus and numerous ramifications are also observed (“7”). Processes of fiber cells are omnipresent. Scale bar 5 μm in a; 4 μm in b; 3 μm in c

Fig. 7

Cell diversity in the marginal zone of H. hongkongensis (H13). a Crystal cells (arrow) were rather scarce and located at the marginal edge of animals under the upper epithelium, sometimes close to the rim. b Observation of a live specimen under DIC illumination illustrates the numerous cilia visible at the edge, shiny spheres of the upper epithelium reaching to the periphery, and a bit toward the central part of the animal disc, the typical birefringent crystals of crystal cells (arrow). c–e At the TEM, cells with a dark cytoplasm, numerous irregularly shaped dark granules, a long neck reaching the surface, and a cilium were frequently observed (asterisk). e some round cells with little cytoplasm might correspond to cells that have recently split (dot). f Atypical cells with a poorly defined nucleus, numerous mitochondria, and large dense-core vesicles are also observed (triangle). At least 6 different morphologically distinct cell types (numbered 1–6) can be distinguished in this figure. Scale bar 6 μm in a; 16 μm in b; 1.5 μm in c–f