The ventral epithelium of Trichoplax adhaerens deploys in distinct patterns cells that secrete digestive enzymes, mucus or diverse neuropeptides

Abstract

The disk-shaped millimeter-sized marine animal, Trichoplax adhaerens, is notable because of its small number of cell types and primitive mode of feeding. It glides on substrates propelled by beating cilia on its lower surface and periodically pauses to feed on underlying microorganisms, which it digests externally. Here, a combination of advanced electron and light microscopic techniques are used to take a closer look at its secretory cell types and their roles in locomotion and feeding. We identify digestive enzymes in lipophils, a cell type implicated in external digestion and distributed uniformly throughout the ventral epithelium except for a narrow zone near its edge. We find three morphologically distinct types of gland cell. The most prevalent contains and secretes mucus, which is shown to be involved in adhesion and gliding. Half of the mucocytes are arrayed in a tight row around the edge of the ventral epithelium while the rest are scattered further inside, in the region containing lipophils. The secretory granules in mucocytes at the edge label with an antibody against a neuropeptide that was reported to arrest ciliary beating during feeding. A second type of gland cell is arrayed in a narrow row just inside the row of mucocytes while a third is located more centrally. Our maps of the positions of the structurally distinct secretory cell types provide a foundation for further characterization of the multiple peptidergic cell types in Trichoplax and the microscopic techniques we introduce provide tools for carrying out these studies.

Conserved biophysical features of the CaV2 presynaptic Ca2+ channel homologue from the early-diverging animal Trichoplax adhaerens

“…At higher magnification, it is apparent that TCaV2 protein is expressed within mucocytes, in small punctate regions adjacent to larger WGA-positive regions (figure 3A inset). The latter likely represents clusters of mucouscontaining vesicles labelled by WGA (23), demarking large cytoplasmic regions of separate mucocyte cells. Pre-incubation of the TCaV2 antibody with the blocking peptide, or staining in the absence of primary antibody, did not produce fluorescent signals above background levels, suggesting that the observed labeling was specific to TCaV2 (figure 3B and C, respectively).…”

Early metazoan origin and multiple losses of a novel clade of RIM pre-synaptic calcium channel scaffolding protein homologues

“…However, only a small number (generally <4) of probe labels were associated with individual nuclei. The small number of probe labels likely indicates that mRNA for these proteins is in low abundance, since much higher probe label densities have been observed by the same in situ hybridization technique with probes for highly expressed proteins, such as digestive enzymes (Mayorova, et al 2019). The higher abundance of Ca V 1 expressing cells near the edge and within the lipophil zone than in the intervening region is interesting because secretory cells are prevalent near the edge and in the lipophil zone but are rare in the intervening region.…”

“…The higher abundance of Ca V 1 expressing cells near the edge and within the lipophil zone than in the intervening region is interesting because secretory cells are prevalent near the edge and in the lipophil zone but are rare in the intervening region. Mucocytes are the most prevalent secretory cell type near the edge and can be recognized by staining with a fluorescence conjugated lectin, wheatgerm agglutinin (WGA) (Mayorova, et al 2019). Combining FISH for calcium channels with WGA staining (Figure S1B) showed that probe labels for Ca V 1 and Ca V 2 were often present inside WGA stained mucocytes, whereas only a few mucocytes had Ca V 3 probe labels in their interiors.…”

“…Average counts of fluorescent granules within regions of the animal characterized by distinct cell-type content (i.e. the edge, fiber cell zone and lipophil cell zone (Mayorova, et al 2019)), revealed that II-RIM is more abundantly expressed than I-RIM overall and in each region (Figure 1F; p-values for Tukey’s tests after one-way ANOVAs: edge <0.05; fiber zone <0.0005; lipophil zone <0.00005; sum of regions <0.00005; ANOVA for separate regions: df = 5, F = 17.1, p = 2.1E-15; ANOVA for regions combined: df = 1, F = 24.5, p = 2.1E-6). Notably, these patterns are consistent with mRNA expression levels measured as average transcripts per million (TPM) in the transcriptome data, where II-RIM is more abundantly expressed than I-RIM at the whole animal level (Figure 1B).…”

“…Trichoplax adhaerens animals were prepared for fluorescence in situ hybridization (FISH) by freezing in tetrahydrafuran (THF) overnight on dry ice followed by fixation in 3% acetic acid in methanol (MeOH) for 30 minutes at −20°C and then 4% paraformaldehyde in methanol for 30 minutes at room temperature, as described (Mayorova, et al 2019). In situ hybridization was performed with RNAscope probes for I-RIM (#72781-C3), II-RIM (#572791-C2), Ca V 1 (#442461), Ca V 2 (#442471) and Ca V 3 (#488711) and Multiplex Fluorescent Assay reagents (#320850) from Advanced Cell Diagnostics (Hayward, CA, USA).…”