Fig 1.
(A) Merged brightfield and fluorescence image of TH1 pausing to feed on Rhodomonas salina microalgae (red particles). The image was captured with a confocal microscope (reproduced from [4]). Cells in the ventral epithelium secreted substances that lysed algae, releasing fluorescent phycoerythrin (red clouds). (B) Phylogenetic tree for Metazoa [11] and phylogenetic relationships among four placozoan species here studied. (C) A drawing of main cell types in TH1 as observed by transmitted electron microscopy (reproduced with permission from [16]). DEC – dorsal epithelial cell; VEC – ventral epithelial cell.
Fig 2.
Fluorescence in situ hybridization (FISH) localization of expression of lipophil and digestive gland cells markers in T. adhaerens H1 wholemounts (A, B) and dissociated cell preparations (C, D).
Images of wholemounts are horizontal (xy) and vertical (xz, from boxed region on xy) maximum intensity projections encompassing ~1/4 of the diameter of the animal (edge of the animal is outlined white). Mucocytes are labeled with WGA. (A) Lipophil cell markers (Ta Tetraspanin, Ta 63996, and Ta GABA transporter) are co-expressed in scattered clusters ~8 µm in diameter throughout the central region of the animal, starting ~ 10 µm from the rim. (B) Digestive cell markers (Ta Trypsin and Ta Chymotrypsin) are highly expressed in a region starting ~60 µm from the rim. Cells expressing a lipophil specific marker (Ta GABA transporter) are interspersed among the digestive gland cells in this region. (C and D) Lipophil cell markers (C, Ta GABA transporter, and D, Ta Tetraspanin) and digestive gland cell markers (C, Ta PLA2, and D, Ta Chymotrypsin) are expressed in different populations of cells. Nuclei are labelled with DAPI (maximum intensity projections merged with DIC). Color separated images corresponding to A–D are shown in S2 Fig. fc – fiber cells; ec – epithelial cells; lc – lipophil cells. Scale bars 10 µm.
Fig 3.
Ultrastructural features of lipophil cells and the structure of a highly expressed lipophil cell secretory protein.
(A) Transmission electron microscopy (TEM) image of a thin section from a lipophil cell (LC) with osmiophilic material in its granules. The inset shows a TEM image of a section from an animal fixed with osmium, which better preserved the lipidic content of the granules and their cores. (B) SEM image of a section taken in a backscatter mode shows that granules in basal portions of LC have a dense core (artificially colored red) surrounded by electron lucent content. Inset is an enlarged view of the boxed region. Arrowhead indicates an apical LC granule. (C) Freeze fracture replica showing a basal part of a LC with multiple granules. Note dual component nature of the granules. A fracture through the interior of a core reveals heterogeneous content resembling cytoplasm. Arrowhead marks a protrusion emanating inward from the granule wall. (D) TEM image of 100 nm thick section showing basal region of a LC as inferred from its proximity to the dorsal side of the animal. Red box depicts a region with a protrusion inside a granule, further studied by EM tomography in (E). (E) Upper panel is an EM projection showing direct connection between the protrusion and the inner surface of the granule. Lower panel is a 1.75 nm thick virtual slice through a reconstructed volume of the tomogram. Note that ER (arrowhead) penetrates the protrusion. (F) TEM image of an apical part of an LC showing exocytosis (arrowhead) of a large granule. Note osmiophilic material and membranous particles located near the site of exocytosis. (G) Freeze fracture replica showing an apical part of a LC and its apical granule. (H) A protein present exclusively in LC secretome is largely composed of alpha helices (AlphaFold per-residue confidence score, pLDDT, color-coded: dark blue > 90 very high confidence; light blue 90 > pLDDT > 70 confident; yellow 70 > pLDDT > 50 low confidence; and orange < 50 very low confidence) and is positively charged (electrostatic map created with ChimeraX). dec – dorsal epithelial cell; e – e-face; fc – fiber cell; lc – lipophil cell; p – p-face. Scale bars 5 μm (B, D), 800 nm (A, C, F, G), and 50 nm (E).
Fig 4.
Two monociliated ventral epithelial cell (VEC) types: digestive gland cells and cells expressing putative antimicrobial peptides (AMPs).
(A) Cells secreting digestive enzymes bear a cilium (combination of FISH for Ta Trypsin and Ta PLA2 and immunolabelling for tubulin; left panel shows fluorescence merged with DIC). (B, C) TEM shows that both peripheral (B) and central (C) ciliated epithelial cells have ~500 nm diameter granules located close to the apical surface. Boxed regions are magnified in insets. (D) The granules in peripheral cells are more electron dense and abundant than those in cells in more central regions. * p<0.05. (E) Trypsin is highly expressed in the central region of the ventral epithelium, while AMP1 expression is restricted to the peripheral region. (F) In dissociated cell preparations, two distinct subpopulations of cells expressing either AMP1 or Ta Trypsin are apparent. Most AMP1 expressing cells strongly express AMP2 whereas Ta Trypsin expressing cells show weak AMP2 expression (not visible in E). A dissociated trypsin+ cell (F, green) contains one red AMP2 grain. (G) Venn diagrams based on cell counts show that three quarters of VEC express either AMP1 or Ta Trypsin along with AMP2. Some mucocytes and DEC express AMP1 with or without either AMP2 or Ta Trypsin. Only a few lipophil and fiber cells express AMP1 or Ta Trypsin. Color coding for Venn diagrams is the same as the fluorescence colors on E and F; double co-expression is indicated as strips of respective colors, triple co-expression is white, and the absence of expression is dark gray. Numbers of counted cells are shown. Scale bars: 10 µm (A, E, F) and 1 µm (B, C).
Fig 5.
Main secretory cells in the dorsal epithelium.
(A) Freeze fracture replica at the apex of a dorsal epithelial cell (DEC) imaged in TEM reveals e- or p-faces of numerous ~500 nm diameter secretory granules. (B, C) TEM of ultrathin sections labelled with nanogold conjugated WGA. Transverse section in the dorsal epithelium (B) shows multiple WGA-stained granules in ciliated DECs; insets show enlarged view of boxed regions. Transverse section at the transition region between dorsal and ventral epithelia (C, the border is demarcated by dotted line) shows that DEC granules bind more WGA than do morphologically similar granules in VEC. (D–H) Confocal images of wholemounts (D, F) and dissociated cell preparations (E, G, H). Many DEC express Ta Intelectin 60661 (D, whole mount; E, dissociated cells) as evident from co-labeling with WGA. Mucocytes (m) label intensely with WGA, but do not express Ta Intelectin 60661. Other cell types (VEC, lipophil (lc) and fiber (fc) cells) are not labeled. (F) Ta ELPE is expressed in nearly all DEC and a few VEC (see xz inset in F and color separated images in S9A Fig); (G, I) About half of Ta ELPE+ co-expresses Ta Intelectin 60661. (H) Some VEC, identified based on their small sizes and cylindrical shapes express Ta ELPE but not Ta Intelectin 60661. (J) Cells co-expressing Ta ELPE and Ta Intelectin 60661 are larger than those expressing only Ta ELPE. e – e face; fc – fiber cells; lc – lipophil cells; m – mucocytes; p – p face.
Fig 6.
Localization and characterization of peptidergic secretory cells.
Trichoplax wholemounts and dissociated cell preparations were labeled with FISH probes for different prepropeptides and other cell-type specific proteins. Mucocytes were labeled with fluorescent-conjugated WGA in wholemounts. Cilia were immunolabeled with an antibody against acetylated tubulin and nuclei were labeled with DAPI in dissociated cell preparations. Circle plots and box plots use the same color coding as fluorescence images; co-expression is indicated as stripes of respective colors. (A) Prepropeptide Ta ELPE (A1) was expressed by DEC throughout the animal and by some VEC near the rim. Interspersed among the Ta ELPE+ DEC were scattered cells that expressed an uncharacterized protein (Ta 63786). Probes for Ta ELPE and Ta 63786 labeled distinct populations of ciliated cells in dissociated cell preparations (A1, circle plot; A2). Ta ELPE+ cells were larger in area than Ta 63786+ cells (A3). (B) Ta LF prepropeptide (B1) was strongly expressed in a row of cells in the dorsal epithelium 5 - 10 µm from the rim and more weakly expressed in scattered cells in the ventral epithelium in a region starting 40 µm from the rim. The yellow channel in the color-merged xy and xz images were dislayed with gamma= 0.54 to enhance the visibility of the weakly labeled cells. The yellow channel is displayed with gamma=1.0 (linear) in the upper xz image. The Ta LF+ ventral epithelial cells co-expressed an astacin-like metalloendopeptidase (Ta 26557; B1, B2), while dorsal Ta LF+ cells did not (B1, B3, B4). Both Ta LF+/Ta 26557+ cells (B2) and LF+/Ta 26557- cells (B3) were small ciliated cells (B5). Circle plot B6 shows that Ta LF+ cells (bright yellow) were distinct from Ta ELPE+ cells (light yellow) and Ta Intelectin 60661+ cells (red stripes). (C) Mucocytes were labeled with fluorescent WGA and a probe for a secreted oligosaccharide binding protein (Ta OligoBP; 63702; C1). The central population of mucocytes co-expressed Ta RWa prepropeptide (C1-3). Both Ta RWa- and Ta RWa+ mucocytes lacked a cilium (C2, tubulin label absent). The rectangular insets show Ta RWa- (top) and Ta RWa+ mucocytes (bottom) without the magenta (Ta OligoBP) fluorescence channel. (D) Ta ELP prepropeptide and an astacin-like metalloendopeptidase (Ta 54934; D1) were co-expressed in a row of cells in the ventral epithelium 15 to 30 µm from the rim. The labeled cells were ciliated (D2, D3), co-expressed both genes (D1-4) and had an area of about 9 µm2 (D5). (E) Probes for Ta SIFGa prepropeptide and for a second uncharacterized secretory protein (Ta 60437) labeled separate populations of cells: Ta SIFGa+ cells were prevalent in the dorsal epithelium of the rim (E1) and more sparsely distributed in the dorsal and ventral epithelium further in the interior; Ta 60437+ cells were in a row 20 to 30 µm from the rim (E1). Both the Ta SIFGa+ and Ta 60437+ cells were ciliated (E2, E3). The Ta 60437+ cells were larger than the Ta SIFGa+ cells (E4,) and larger than Ta ELP+/Ta 54934+ cells (E4, red), which were in the same area (D1). (F) A subset of Ta SIFGa+ cells in the ventral epithelium >10 µm from the rim expressed the Ta FFNP prepropeptide (F1, F2). The yellow channel was displayed with gamma=0.75 to enhance the visibility of weakly stained cells. Color separated images displayed with gamma=1.0 (linear) are shown in S9B Fig. Both Ta SIFGa+/Ta FFNP- cells (F3) and Ta SIFGa+/Ta FFNP+ cells (F4, F5) bear a cilium. The Ta SIFGa+/Ta FFNP+ cells were larger than the Ta SIFGa+/Ta FFNP- cells (F6). * p<0.05. Scale bars 20 µm (A1, B1, F1), 10 µm (C1, D1, E1) and 5 µm in all dissociated cells images.
Fig 7.
Localization and characterization of peptidergic cells.
(A) Trichoplax wholemount (top panel, horizontal maximum intensity projection; bottom panels, vertical projections of boxed region). Mucocytes were labeled with fluorescent WGA. Cells co-expressing Ta LF and astacin Ta 26557 are interspersed among mucocytes and Ta PLA2+ cells in the central part of the ventral epithelium while cells that express Ta LF prepropeptide without astacin are most prevalent close to the rim. (B) Dissociated cell preparation. Nuclei were labeled with DAPI. Color separated and merged views show multiple cells that co-express Ta LF and astacin Ta 26557 (yellow arrowheads), a cell that expresses astacin without Ta LF (magenta arrowhead) and cells that express Ta PLA2 (white arrowheads). (C) Many cells co-express Ta LF and astacin Ta 26557, but few cells co-express Ta LF+ or Ta 26557 and Ta PLA2. (D) All labeled cells are similar in size with Ta PLA2+/Ta 26557+ cells slightly larger. (E) Horizontal and vertical maximum intensity projections of a wholemount labeled with probes for Ta LF prepropeptide, secretory protein Ta 63786, putative antimicrobial peptide AMP4 and fluorescent WGA. The images span nearly the entire width of the animal – note the mucocyte and the Ta LF+ cell at the upper right. Cells in a region of the ventral epithelium within 10 to 40 µm of the rim co-express Ta 63786 and AMP4; cells in the peripheral part of the dorsal epithelium express Ta LF or Ta 63786 but do not express AMP4. (F) Enlarged view of partially dispersed cells co-expressing Ta 63786 and AMP4. (G) Horizontal and vertical maximum intensity projections of an animal labeled with probes for Ta LF, Ta WPPF prepropeptides, AMP2, and the lectin WGA. The images encompass ~1/3 the diameter of the animal. Cells expressing Ta WPPF prepropeptide are distributed in the dorsal epithelium in a region starting ~40 μm from the rim and do not express Ta LF or AMP2. Scale bars 20 µm in whole mount images and 5 µm in dissociated cells images.
Fig 8.
Secretory behaviors of Trichoplax associated with external digestion of algae.
(A–E) Confocal images of a Trichoplax feeding on R. salina algae; (F, G) normalized fluorescence intensity measured in selected regions. A starved animal was transferred to a cover glass chamber containing algae in seawater with a lipophilic dye (LipidTOX, red) that stains lipophil cell granules; FM1-43 (cyan), a membrane dye used here to visualize the secreted contents of lipophil cell granules; and a fluorescent indicator for trypsin activity (BZiPAR, green). The insets numbered with lowercase letters are enlarged view of rectangular region on a respective image numbered with an uppercase letter. (A, a) At t=-1.9 sec, the animal had ceased moving in a region containing algae (blue, phycoerythrin autofluorescence, arrows) and debris (cyan) representing algae remnants from an earlier feeding episode. The animal body is outlined white, and the feeding pocket is outlined red. (B, b) At t=0 sec, lipophil cell granule secretion was evident in the feeding pocket due to the sudden appearance of small (<5 µm) diffuse clouds and bright particles (arrowheads on b) of FM1-43-stained material. (C, c) At 5.7 sec, some algae near sites of lipophil granule secretion swelled, lysed, and became intensely stained with LipidTOX and FM1-43 (pink in merged, arrows in c). Other algae (blue) remained intact and were not stained with LipidTOX or FM1-43. (D, d) By 195.4 sec, diffuse BZiPAR fluorescence (green) filled the feeding pocket. The lysed algae no longer were visible, but the intact algae remained (arrows in d). (E) Intensity encoded BZiPAR fluorescence images show increasing trypsin activity between 70.6 and 233.5 sec. (F) Details of first events in feeding: Lipophil granules (cyan; the fluorescence profile obtained for the area outlined yellow in b) were secreted approximately synchronously at 0 sec and this was followed by a rapid increase of fluorescence in nearby algae (four fluorescence profiles obtained for four pink algal cells in c; different shades of magenta). (G) Evidence of digestion: algae affected by lipophil granules (different shades of magenta) burst and released their content. Secretion of trypsin, as indicated by BZiPAR fluorescence (green; fluorescence profile obtained for the region outlined red in B), began 40–50 sec after lipophil discharge and was associated with a decline of fluorescence intensity of the lysed algal cells. Those algae not affected by lipophil granules remained constant in intensity throughout the feeding episode (two fluorescence profiles obtained for two individual algal cells; different shades of blue). Scale bars 100 µm.
Fig 9.
Summary diagrams of distributions of Trichoplax adhaerens secretory cell types.
(A) Secretory proteins, expressed specifically in DEC, pVEC, cVEC, mucocytes, and lipophil cells (peptidergic cells represented in panel B are omitted in panel A). Many central DEC co-express Ta Intelectin and the precursor of ELPE peptide but peripheral DEC do not express Ta Intelectin. Peripheral VEC highly express precursors of putative antimicrobial peptides (AMP’s), while cVEC highly express digestive enzymes. Lipophil cells and mucocytes differentially express their own specific genes (see legend). (B) Secretory products of peptidergic cell types. Localization of peptidergic cell types is based on measurements from animals labeled with FISH probes for peptide precursors (Figs 6, 7). Peptidergic cells located in the central zone of the animal co-express additional secretory products unlike the cells located at the periphery. Only mucocytes in the central zone co-express TaRWa. Diagrams represent vertical cross sections of a region encompassing ~20% of the diameter of an animal ~500 µm in diameter. Cell dimensions are based on measurements from animals prepared for transmission electron microscopy by high pressure freezing and freeze substitution [16].
Table 1.
RNAscope probes for multiplex fluorescence in situ hybridization.