Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis

Abstract

Introduction: Nematostella vectensis, a member of the cnidarian class Anthozoa, has been established as a promising model system in developmental biology, but while information about the genetic regulation of embryonic development is rapidly increasing, little is known about the cellular organization of the various cell types in the adult. Here, we studied the anatomy and development of the muscular system of N. vectensis to obtain further insights into the evolution of muscle cells.

Results: The muscular system of N. vectensis is comprised of five distinct muscle groups, which are differentiated into a tentacle and a body column system. Both systems house longitudinal as well as circular portions. With the exception of the ectodermal tentacle longitudinal muscle, all muscle groups are of endodermal origin. The shape and epithelial organization of muscle cells vary considerably between different muscle groups. Ring muscle cells are formed as epitheliomuscular cells in which the myofilaments are housed in the basal part of the cell, while the apical part is connected to neighboring cells by apical cell-cell junctions. In the longitudinal muscles of the column, the muscular part at the basal side is connected to the apical part by a long and narrow cytoplasmic bridge. The organization of these cells, however, remains epitheliomuscular. A third type of muscle cell is represented in the longitudinal muscle of the tentacle. Using transgenic animals we show that the apical cell-cell junctions are lost during differentiation, resulting in a detachment of the muscle cells to a basiepithelial position. These muscle cells are still located within the epithelium and outside of the basal matrix, therefore constituting basiepithelial myocytes. We demonstrate that all muscle cells, including the longitudinal basiepithelial muscle cells of the tentacle, initially differentiate from regular epithelial cells before they alter their epithelial organisation.

Conclusions: A wide range of different muscle cell morphologies can already be found in a single animal. This suggests how a transition from an epithelially organized muscle system to a mesenchymal could have occurred. Our study on N. vectensis provides new insights into the organisation of a muscle system in a non-bilaterian organism.

Figure 1

The muscle system of N. vectensis. Schematic overview of the muscle system in the adult polyp. The columnar musculature consists of the longitudinally orientated parietal and retractor muscle located in each of the eight mesenteries and a columnar ring muscle: All are of endodermal origin, the latter spanning the whole column between parietal muscles. In the tentacle an epidermal longitudinal muscle antagonises a gastrodermal ring muscle. Corresponding cross-sections of the polyp through the pharynx (1) and the subpharyngeal (2) region are shown to demonstrate the internal bilateral symmetry based on the arrangement of retractor muscles. crm, column ring muscle; gc, gastric cavity; m, mesentery; pam, parietal muscle; ph, pharynx; rem, retractor muscle; t, tentacle; tlm, tentacle longitudinal muscle; trm, tentacle ring muscle.

Figure 2

Histology of the muscle system of N. vectensis. All sections are cross-sections. A Architecture of a single mesentery. The parietal muscle is located at the base of the mesentery, followed by a more distal retractor. Between the retractor muscle and the mesenterial filament at the tip of the mesentery, a gonad, embedded into the mesoglea, is formed. Inlet: Cross-section through the subpharyngeal region showing all eight mesenteries. B Detail of A. The parietal muscle shows a typical bilateral folding of the mesoglea, on which the myonemes of the muscle cells attach. The muscle consists of two contiguous sheets of myonemes, which merge into the ring muscle laterally. Two distinct neural plexi are visible on either side next to the parietal muscle. C Detail of A. In the retractor muscle the mesoglea folds and branches only to one side. This folding is generally more pronounced than in the parietal muscle. D Detail of A. The ring muscle lies between the parietal muscle of each mesentery. Similarly, the myonemes are located at the base of the cells. ep, epidermis; gd, gastrodermis; go, gonad; mef, mesenterial filament; mg, mesoglea; myo, myoneme; np, neural plexus; pam, parietal muscle; rem, retractor muscle; crm, column ring muscle; s, stalk. Scale: A, C: 100 μm; B, D: 50 μm; inlet: 1 mm. All sections are stained with Azan.

Figure 3

Muscles of the adult polyp revealed by F-actin staining with phalloidin. A Cross-section of a whole mesentery with proximal parietal muscle and distal retractor muscle. B Detail of A. Parietal muscle. Note the transition of the columnar circular muscle (arrows) into the longitudinally orientated parietal muscle (arrowhead). C Detail of A. Retractor muscle (arrow). D Cross-section of a tentacle. E Detail of D. The well-formed longitudinal muscle is located basiepithelially in the epidermis (arrowhead), whereas the circular muscle is weakly developed and located in the gastrodermis (arrow). F Single longitudinal optical section of a tentacle showing the strong longitudinal muscle filaments (arrowhead) overlying those of the circular muscle (arrow). white: DAPI, orange: phalloidin.

Figure 4

Epithelial organization of the columnar muscles. All sections are cross-sections A Overview of the retractor muscle region. B Overview of the parietal muscle region. C Detail of B. A distinct neural plexus consisting of numerous neurites is located next to the parietal muscle and close to the transition zone to the ring muscle. Inlet 1: spot-like adherens junctions between the basal part (black arrow) connect muscle cells to each other. Focal adhesions (white arrow) connect the cells to the mesoglea. Inlet 2: Detail image of neurites containing neural vesicles (white arrow). D Detail of B. Parietal muscle cells consist of a cell body involved in building up the epithelium. Via cytoplasmic bridges they are connected to the myofilament-containing basal part of the cell, adjacent to the mesoglea (cell highlighted in yellow); this yields a consistent sheet of myonemes. E and F Details of A. Muscle cells (highlighted in yellow) remain epithelial at least in the proximal and distal boundaries of the retractor muscle. E (inlet): Focal adhesion between muscle cell and mesoglea. G Detailed image of well-formed myonemes in the central region of the retractor muscle. Thin cytoplasmic bridges (inlet, black arrow) are projected by every cell. Basal parts of the cells are connected by spot-like adherens junctions (black arrows). H Detailed image of a ring muscle cell showing its participation in building up the epithelium. The myofilaments are located at the basal part of the cell. cb, cytoplasmic bridges; gc, gastric cavity; gd, gastrodermis; m, mitochondrium; mg, mesoglea; myo, myoneme; np, neural plexus; nu, nucleus; pam, parietal muscle; rim, ring muscle. Scale: A, B: 20 μm, C-F, H: 5 μm, G: 2 μm.

Figure 5

Organization of the tentacle muscle system in an adult polyp. A Tentacle cross-section. B Detail of A. Typical appearance of a tentacle epithelium. Longitudinally orientated muscle cells are situated side by side at the base of the epidermis and are connected to the mesoglea (highlighted in red). A neural plexus (highlighted in green) is located on top of the muscle layer. Epithelial cells are still connected to the mesoglea by thin processes, which find their way through the discontinuous sheet of muscle cells. In the gastrodermis the epithelial muscle cells are oriented circularly. C Longitudinal section of a longitudinal muscle cell pointing out the basiepithelial organization. Note the close location of the nucleus to the myofilaments. Inlet: parallel arrangement of thick myofilaments (arrow). D Detail of a cross-sectioned longitudinal muscle cell with adjacent nucleus. White arrow: spot-like adherens junction connecting muscle cells to each other. Inlet: Thick myofilaments (black arrow) are distributed irregularly between thin ones. E Ring muscle cell of the gastrodermis (cross-section) with myofilaments located at the base of the cell adjacent to the mesoglea. White arrow: Belt-like apical junctional complex. Inlet: In comparison to the longitudinal muscle the myofilaments of the ring muscles are weakly developed. Black arrow: Thick myofilament. F Epithelial cell (highlighted in yellow) spanning from the external surface to the mesoglea through the sheet of longitudinal muscle cells. Asterisk denotes mesoglea. c, cilium; ed, epidermis; epc, epithelial cell; gc gastric cavity; gd; gastrodermis; lm, longitudinal muscle layer, lmc, longitudinal muscle cell; mv, microvilli; np, neural plexus; nu, nucleus; muscle; rm, ring muscle layer; s, spirocyst; t1, type 1 gland cell with electron-light vesicles; t2, type 2 gland cell with electron-dense vesicles. Scale: A: 50 μm, B: 5 μm, C-F: 2 μm.

Figure 6

Development of columnar longitudinal muscles in mid and late planula. A Mid-planula (~4 d), overview. The first two emerging mesenteries (primary mesenteries) are formed opposite each other. Note that the location of the secondary mesenteries is already set at this stage (inlet). B Detail of A. The base of the mesentery (corresponding to the future site of parietal muscle formation) shows no distinct accumulation of myofilaments. C Detail of A. Single cell (inlet, black arrow: myofilaments) showing first sign of muscle formation. D Late planula (~5 d), overview. Primary mesenteries gradually shift to one side. E Detail of D. The developing retractor muscle in the primary mesentery can already be identified. Myonemes are formed exclusively on one side of the mesoglea. Black arrows: accumulating myofilaments. F Detail of D. Basal part of the primary mesentery. No myofilaments have accumulated yet. Note an amoeboid cell at the branching of the mesoglea, which could be detected occasionally (asterisk). G Detail of D. Secondary mesentery. Future retractor muscle cell highlighted in red. H Detail of G. Retractor muscle cells forming at the tip of the mesentery (in contrast to the primary mesentery), having no bias to one side at this stage. Black arrows: accumulating myofilaments. I Detail of G. Cells at both sides of the base of the mesentery start to accumulate myofilaments (black arrows) adjacent to the mesoglea. All sections are cross-sections of the subpharyngeal region. For easier understanding the mesoglea of all close-ups is highlighted in yellow. Inlets in A and D indicate the location of primary (lines with circles) and secondary mesenteries (lines without circles). ec, ectoderm; en, endoderm; gc, gastric cavity; pm, primary mesentery. Scale: A, D: 50 μm; B, C, G: 5 μm; E, F: 2 μm; H, I: 1 μm.

Figure 7

Development of columnar longitudinal muscles in primary polyps and juveniles. A Early primary polyp (~5 d), overview. B Detail of A. Cells located next to each other at the base of the mesentery increasingly accumulate myofilaments (black arrows) in their basal part. C Detail of A. Some retractor muscle cells (long black arrows) start to constrict the basal from the apical part of the cell, leaving behind thin cytoplasmic bridges (short black arrows), while in some the nucleus still lies near the myoneme (short white arrow). D Detail of A. Secondary mesentery. E Detail of D. As in primary mesenteries more cells accumulate myofilaments (black arrows) at their bases. F Detail of D. In the secondary mesenteries, myonemes (black arrows) are still located at the tip of the mesentery, but are more pronounced on one side. G Juvenile (~3 months) polyp, overview. H Detail of G. Despite the constriction of the myoneme, parietal muscle cells retain an epithelial organization (single cell highlighted in red). Inlet: Arrangement of myofilaments and adherens junctions between myonemes. I Detail of G. In the retractor muscle the sheet of myonemes starts to fold. Muscle cells are still epithelial (single cell highlighted in red). Inlet: Arrangement of myofilaments and adherens junctions between myonemes. J Optical cross-section of a mid-planula. The orientation of the retractor muscle is species-specific and predetermined for every mesentery. This allows predicting the future side of the retractor muscle in all mesenteries, as soon as all mesenteries have emerged. Red arrows: primary mesenteries, white arrows: secondary mesenteries. ec, ectoderm; en, endoderm; gc, gastric cavity; pb, proximal bulge; pm, primary mesentery; sm, secondary mesentery; mf, mesenterial filament, myo, myoneme; np, neural plexus; nu, nucleus. Scale: A, G: 50 μm; B, C: 2 μm; D, H, I: 5 μm, E: 500 nm; F: 1 μm.

Figure 8

Condensation of actin filaments during the differentiation of muscle cells. A, D, G: Early planula. F-actin is weakly pronounced in the mesenteries but not orientated uniformly. B, E, H: In the late planula, F-actin becomes increasingly accumulated and oriented along the oral-aboral axis adjacent to the mesoglea. Moreover, future tentacle cells become enriched in F-actin (B, inlet 1). C, F, I: Early primary polyps show distinct longitudinal muscle strands and increased F-actin accumulation in tentacle muscle cells (C, inlet 1). The parietal muscle and retractor muscle can clearly be identified. Images in A (6 sections), B (8 sections) and C (20 sections) constitute maximum projections. pam, parietal muscle; rem, retractor muscle.

Figure 9

Differentiation of tentacle longitudinal muscle cells in a muscle specific transgenic (MHC::mCherry) reporter line. F-actin was stained with phalloidin (green), mCherry with an α-RFP antibody (red). A-G Optical longitudinal sections of mid-planula stages (4–5 d) in the future tentacle bulb region. A Overview. B-D Maximum projection of six layers. An early undifferentiated tentacle muscle cell (labelled by the α-RFP antibody) spans throughout the ectodermal epithelium and already shows a weak accumulation of actin filaments (D, inlet). E-G Maximum projection of seven layers. Tentacle muscle cells lose connection to the apex of the epithelium. Actin filament accumulation at the base of the epithelium increases in several cells (G, arrows). H-N Optical longitudinal sections of early primary polyp stages (7 d). Maximum projection of 21 layers. Different stages of detachment of tentacle longitudinal muscles from the epithelium co-occur at the same stage (J, long arrow: epithelial muscle cell, short arrow: submerged muscle cell). Actin filaments accumulate in several cells before mCherry becomes visible (K, arrows). L-N Columnar retractor muscle cells expressing mCherry. O-Q Optical longitudinal sections of primary polyps (13 d). Maximum projection of 53 layers. Numerous actin filaments are enriched in the outgrowing tentacle bulbs (Q, arrow). Tentacle longitudinal muscles have lost their connection to the apex of the epithelium and become situated at the base of the ectoderm, increasing their length with tentacle elongation (P, arrow).

Figure 10

Schematic representation of organization and development of muscle cells. A Modes of muscle cell organization. The type 1 muscle cell corresponds to the classical epitheliomuscular cell. The cell is integrated into the epithelium by apical cell-cell junctions, connecting them to neighboring cells, while the basal part is elongated and houses myofilaments. Type 2 muscle cells are still epitheliomuscular; their connections between the apical and the basal part, however, are drawn out to form thin and elongated cytoplasmatic bridges. In type 3 muscle cells the apical cell junctions have been lost, resulting in a sinking to the base of the epithelium, with the nucleus situated near the myofilaments. B + C Development of type 2 & 3 muscle cells. In both types myofilaments start to become accumulated at the base of epithelial cells. B In type 2 muscle cells the apical cell-cell junctions remain and the connection between the apex and the base becomes constricted to form thin and elongated cytoplasmic bridges. The basally situated myonemes build up a continuous and folded muscle sheet. C Type 3 muscle cells lose their apical cell-cell junctions and sink down to the base of the epithelium, where they are arranged in a discontinuous muscle layer, enabling overlying epithelial cells to attach to the mesoglea. Color code: yellow: mesoglea, blue: epithelium, red: myofilaments, grey, nucleus.