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Figure 1. Overview of musculature of Ophryotrocha diadema. Scale bars = 50 μm, anterior is to the left. A. Dorsal view, phalloidin staining. The most prominent muscles in the prostomium are the ventral diagonal muscles (dVLM). Two transverse muscles are present in the prostomium: PStm2 crosses the dVLM dorsally, whereas PStm1 lies between the dorsal and the ventral longitudinal muscles. The complex musculature of the ventrally situated pharynx (phar) is heavily stained. The inner strands of the dorsal longitudinal muscles (DLM) cross repeatedly at the dorsal midline (circles). Four pairs of parapodia are present, with prominent parapodial muscle complexes (pmc). Transverse (tm) and diagonal muscles (dm) cross the dorsal side irregularly. Several transverse muscles surround the posterior zone of segment proliferation (arrowhead). The last transverse muscle circle marks the pygidium (tagged arrow). B. Ventral view, phalloidin staining (red) and anti-acetylated-tubulin staining (green). The ciliary bands are stained by anti-acetylated tubulin antibody. These are the prototroch (prot) of the prostomium, the metatroch (met) and the ciliary band of the segment like annulus within the peristomium (pcb), and the four ciliary bands of the chaetigers (1–4 BS) anterior to the telotroch (tel) of the pygidium. The boxed region marks the close-up depicted in Figure C (vnc-ventral nerve cord, VLM-ventral longitudinal muscle) C. Close-up from B. Ventral view, phalloidin staining and anti-acetylated tubulin antibody depth coded (in μm). Upper panel shows the ventral nerve cord (vnc), lower panel shows the median ventral longitudinal muscle (mVLM). Depth coding indicates that the mVLM is situated dorsal (deep) to the vnc (circles). D. Histological cross section through chaetiger of an adult. The dorsal longitudinal muscles (DLM) lie just below the epidermis (epi). The uniramous parapodia (ppd) are ventrolaterally situated. The ventral longitudinal muscles (VLM) lie on either side of the ventral nerve cord (vnc). E. Close up from D. Same labeling as in D, additionally labeled are the dorsally situated transverse muscles (tm). F. Close up from E depicting the dorsal longitudinal muscle (DLM) and a transverse muscle (tm) lying above it (supralongitudinally). G. 3D-reconstruction from a series of histological sections in the same body-region as shown in D. Anterior is toward the top of the figure. Depicted are the dorsal longitudinal muscles (DLM) with their dorsomedian crossing strands (arrow), diagonal muscles (dm) and transverse muscles (tm) in a supralongitudinal position. The ventral longitudinal muscles (VLM) and the median ventral longitudinal muscle (mVLM) surround the ventral nerve cord (vnc – only the main tracts of the neuropile shown here). The anterior and posterior dorsal parapodial muscles (adPM, pdPM) stretch diagonally toward posterior and anterior respectively, underneath the dorsal longitudinal muscles.
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Figure 2. Prostomial muscles. Scale bars = 50 μm, anterior is to the left. A. Adult specimen with ten chaetigers: ventral view, phalloidin staining. Inset shows phalloidin staining (red) and anti-acetylated tubulin staining (green). Frames mark the regions used for 3D-reconstruction in panels B-E and Figure 3. Pharynx (phar), ventral longitudinal muscle (VLM) and median ventral longitudinal muscle (mVLM). B. Close up of frame depicted in A, phalloidin staining, depth coded (in μm). The prominent straight ventral longitudinal muscles (sVLM) and the diagonal ventral longitudinal muscles (dVLM) connect anteriorly with the outer dorsal longitudinal muscles (oDLM). A small muscle strand splits from each dVLM muscle and extends ventroanteriorly (circle). The most central pair of longitudinal muscle strands cross each other and run toward the opposite lateral side of the prostomium (cDLM). A transverse muscle (PStm1) lies between the dorsal and ventral longitudinal muscles. One part of the PStm1 reaches into the antennae (square). PStm2 – prostomial transverse muscle 2. C – E. 3D-reconstruction of muscles from the area depicted in Figure B. Labels are as follows: sVLM – straight ventral longitudinal muscle, dVLM – diagonal ventral longitudinal muscle, with ventroanteriorly running muscle portion (circle), PStm1 – prostomial transverse muscle 1, contributing to the antennae (square), PStm2 – prostomial transverse muscle 2, lying dorsal to the longitudinal muscles, oDLM, mDLM, iDLM – outer, middle, and inner straight-running branches of dorsal longitudinal muscle, cDLM – central pair of dorsal longitudinal branches, crossing each other. C. Ventral view. D. Ventroanterior view. E. Dorsal view.
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Figure 3. Parapodial muscles. Scale bars = 50 μm, anterior is to the left. A. Ventral view, close up from Fig. 2A, phalloidin staining, depth coded (in μm). Shown are two parapodia and their muscle complexes (pmc). The ventral longitudinal muscles (VLM) lie laterally, and the median ventral longitudinal muscle (mVLM) runs along the midline. Parapodial muscles are only labeled on the right half of the body. These include chaetal muscles (chm); acicular muscles (am); inner anterior parapodial muscle (iaPM); outer anterior parapodial muscle (oaPM); inner posterior parapodial muscle (ipPM); outer posterior parapodial muscle (opPM); median ventral longitudinal muscle (mVLM); the ipPM, opPM, iaPM assemble with the mVLM (circle). Thin transverse muscle strands (tm) cross the ventral side. B. 3D-reconstruction of A, but from dorsal side, same labeling scheme. Each muscle is displayed in a unique color. The dorsal longitudinal muscle (DLM) and the anterior (adPM) and posterior dorsal parapodial muscles (pdPM) have been removed from the reconstruction of the right side of the body, to expose underlying muscles. Chaetal (chm) and acicular muscles (am) form the center of the parapodium. The outer anterior parapodial muscle (oaPM) stretches anteriorly, terminating in the vicinity of the VLM. The inner anterior parapodial muscle crosses the body-cavity and ends at the opposite side (iaPM). Both posterior parapodial muscles (ipPM, opPM) terminate at the midline in close proximity to the median ventral longitudinal muscle (mVLM). Here, the ipPM, opPM, iaPM of two adjoining pairs of parapodial muscle complexes assemble (circle). C. 3D-reconstruction of A, dorsal view, with still more overlying muscles removed to expose ventral musculature. The consecutive parapodial muscle complexes of the right body-half are shown. Labeling as in B. Three muscles, originating from either of the ventral longitudinal muscles (VLM, mVLM) stretch ventroanteriorly (asterisks). Single transverse muscle strands (tm) of the ventral side are visible. D. Same 3D-reconstruction as in C. Ventral view.
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Figure 4. Onset of muscle formation: early first chaetiger ciliary band. Scale bars = 50 μm, anterior is to the left. A. Ventral view, phalloidin staining (red) and anti-acetylated tubulin staining (green). The anlage of the brain (arrow) and the circumoesophageal connectives surround the mouth opening (stom). Prototroch (prot), metatroch (met), the peristomial (pcb) and the first chaetiger ciliary band (1BS) and the telotroch (tel) are stained by the anti-acetylated tubulin antibody. The square marks the region of the close up depicted in C. B. Same picture as A, but only phalloidin staining. At this stage, phalloidin prominently labels the F-actin of the cytoskeleton, marking all cell margins. C. Close up from A, phalloidin staining only. The first discernable muscle fibers of the ventral longitudinal muscle (VLM) become apparent.
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Figure 5. First chaetiger ciliary band – later stage. Scale bars = 50 μm, anterior is to the left. A, C, F. Phalloidin staining shown in red anti-acetylated tubulin staining in green, and nuclear staining in blue. Prototroch (prot), metatroch (met), ciliary band of peristomium (pcb), ciliary bands of chaetigers (BS) and telotroch (tel). Additional labeling as in B, E, H. Phalloidin staining. D, G. Phalloidin staining, depth coded (in μm). A. Same embryo as in B. Ventral view. The ventral longitudinal muscles (VLM) have elongated towards the pygidium. The first transverse muscles (arrow) differentiate. Mouth region (stom) clearly visible. C, D. Same Embryo as in E. Ventrolateral view. The dorsal (DLM) and ventral longitudinal muscles (VLM) have reached the pygidium. Adjacent to the autofluorescent chaetae (ch) lie transverse muscles, the anlage of the paraopodial muscles, laterally and ventrally (arrows). The ventral longitudinal muscles extend into the prostomium, forming three projections (dVLM, asterisk). The jaw apparatus (ja) is apparent ventral to the mouth opening (stom). F, G. Same Embryo as in H. Ventrolateral view. One transverse parapodial muscle anlage (arrow) is always present just anterior and just posterior of a ciliary band (pcb, BS). DLM-dorsal longitudinal muscle, VLM-ventral longitudinal muscle, ch-chaetae, stom-mouth opening.
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Figure 6. Two chaetigerous ciliary bands. Scale bars = 50 μm, anterior is to the left. A-E. Phalloidin staining (red) and in A, D anti-acetylated tubulin staining (green). Prototroch (prot), metatroch (met), ciliary band of peristomium (pcb), ciliary bands of chaetigers (BS) and telotroch (tel). A. Ventral view. Square marks region depicted in B. Labeling as in C. B. Close-up from A, ventral view, phalloidin staining, depth coded (in μm). The parapodial muscle anlage (pma) adjacent to the ciliary bands lie sublongitudinally, i.e., internal to the ventral longitudinal muscle (VLM). C. Same embryo as in A. Ventral view, phalloidin staining. The median ventral longitudinal muscle has now formed (mVLM). Its anterior splits, with branches running around each side of the pharyngeal muscles (phar). The ventral longitudinal muscles (VLM) project into the prostomium as the diagonal ventral longitudinal muscles (dVLM) and the straight ventral longitudinal muscles (sVLM) D. Lateral view. The parapodial muscle anlage (pma) runs on the lateral side of the embryo towards posterior. Each ciliary band (BS) lies between a set of parapodial muscle anlagen (pma). Autofluorescent chaetae (ch) are visible. E. Same embryo as in D. Lateral view, phalloidin staining only. The parapodial muscle anlagen (pma) adjacent to ciliary bands span from the ventral longitudinal muscle (VLM) dorsally to the dorsal longitudinal muscle (DLM). The musculature of the pharynx (phar) exhibits a basket-like shape.
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Figure 7. Three to four chaetigerous ciliary bands: ventral view. Scale bars = 50 μm, anterior is to the left. A-D. Phalloidin staining (inset of A shows phalloidin in red, and anti-acetylated tubulin staining in green) A. Ventral view. The ventral straight (sVLM) and diagonal prostomial muscles (dVLM) reach into the prostomium. The inner projection of the dVLM (asterisk) is less developed than the lateral projections. The pharyngeal muscles (phar) are heavily stained. The muscles of the first three parapodia (forming sequentially) are evident, and illustrate the developmental sequence. The most posterior parapodium shows only the outer posterior parapodial muscle (opPM) at this point, whereas the parapodia of the first chaetiger display the complete adult musculature: outer (oaPM) and inner anterior parapodial muscles (iaPM), chaetal muscles (chm), acicular muscles (am). The outer (opPM) and inner posterior parapodial muscle (ipPM) are not individually discernable. The chaetae of the fourth chaetiger (ch) are already visible. B. Ventrolateral view, phalloidin staining. Labeling as in A. Dorsal longitudinal muscle (DLM). Between this segment and the transverse muscle ring of the pygidium (tagged arrow) several transverse muscles are present within the zone of segment proliferation (open arrow). Ventral longitudinal muscle (VLM), median ventral longitudinal muscle (mVLM). C. Same as in B, phalloidin staining, depth coded (in μm). D. Close up from C, phalloidin staining, depth coded. Muscles of the parapodium: outer (oaPM) and inner anterior parapodial muscle), outer/inner posterior parapodial muscle (opPM), chaetal muscle (chm), acicular muscle (am) and ventral longitudinal muscle (VLM). Chaetal (chm) and acicular muscles (am) are still in close proximity.
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Figure 8. Three to four chaetigerous ciliary bands: dorsal and lateral view. Scale bars = 50 μm, anterior is to the left. A-F. Phallodin staining (B, E also include anti-acetylated tubulin staining in green and nuclear staining in blue). Prototroch (prot), metatroch (met), ciliary band of peristomium (pcb), ciliary band of chaetigers (BS) and telotroch (tel). C, F. Depth coded (in μm). A-C. Lateral view. In addition to the diagonal ventral longitudinal muscle (dVLM), the outer dorsal longitudinal muscle (oDLM) now extends into the prostomium. The pharynx (phar) is ventrally situated. The ventral longitudinal muscle (VLM) elongates straight posteriorly, whereas inner branches of the dorsal longitudinal muscle (DLM) run dorsomedially toward the midline. The arrowhead marks a circle of transverse muscle anterior to the pygidium. D-F. Dorsal view. Anteriorly, the outer dorsal longitudinal muscles (oDLM) and the ventral diagonal muscle (dVLM) connect within the prostomium, although the median projection of the dVLM (asterisk) remains weakly developed. Inner branches of the dorsal longitudinal muscle (DLM) intersect at the dorsal midline (circle). The anterior (adPM) and posterior dorsal parapodial muscles (pdPM) extend from the dorsal side into the parapodial muscle complex (pmc). The arrowhead marks the transverse muscle circle within the region anterior to the pygidium.
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