The evolutionary origin of bilaterian smooth and striated myocytes

Figure 1.. Ultrastructure and core regulatory complexes of myocyte types. (A) Schematic smooth and striated ultrastructures. Electron-dense granules called ‘dense bodies’ separate adjacent myofibrils. Dense bodies are scattered in smooth muscles, but aligned in striated muscles to form Z lines. (B–D) Core regulatory complexes (CoRCs) of transcription factors for the differentiation of different types of myocytes in vertebrates. Complexes composition from (Creemers et al., 2006; Meadows et al., 2008; Molkentin et al., 1995) for skeletal myocytes, (Hoggatt et al., 2013; Nishida et al., 2002; Phiel et al., 2001) for smooth myocytes and (Durocher et al., 1997; Lee et al., 1998) for cardiomyocytes. Target genes from (Blais et al., 2005) for skeletal myocytes, (Nishida et al., 2002) from smooth myocytes and (Schlesinger et al., 2011) for cardiomyocytes. DOI: http://dx.doi.org/10.7554/eLife.19607.002

Figure 2.. Development and ultrastructure of visceral and somatic musculature in Platynereis larvae and juveniles. (A) Development of visceral musculature. All panels are 3D renderings of rhodamine-phalloidin staining imaged by confocal microscopy. Visceral muscles have been manually colored green and somatic muscle red. Scale bar: 50 μm. (B) Schematic of the musculature of a late nectochaete (six dpf) larva. Body outline modified from (Fischer et al., 2010). Ventral view, anterior is up. (C–M) Electron micrographs of the main muscle groups depicted in B. Each muscle group is shown sectioned parallel to its long axis, so in the plane of its myofilaments. Scale bar: 2 μm. (C’,E’) are schematic drawings of the cells shown in (C,E). The Z-lines are made of aligned dense bodies (in black), myofilaments are in red, cytoplasm is in yellow and plasma membrane in grey. Attachment points of myofilaments on the dense bodies are represented with dotted lines when they are outside of the plane of section in the electron micrograph. Zoom panel in C’ shows oblique striation with a 5° angle between myofilaments and Z-lines (compare to Figure 1A). (H) shows another cross-section in the stomodeum of the…

Figure 2—figure supplement 1.. Gut patterning in Platynereis six dpf larvae. (A) six dpf Platynereis larva stained with phalloidin and DAPI to show tripartite gut organization. Maximal Z-projection of a confocal stack, ventral view, anterior side up. The plane of the cross-sections of Figure 2—figure supplement 2 (in slightly older individuals of otherwise similar morphology) is indicated by the dotted line. (B–E) WMISH for gut markers. (B) Ventral view, anterior is up. (C–E) Left lateral views, anterior is up, ventral is right. (F) Schematic of gut patterning in Platynereis late nectochaete larvae. Asterisk is the mouth on all panels. Scale bar: 50 μm. DOI: http://dx.doi.org/10.7554/eLife.19607.004

Figure 2—figure supplement 2.. Formation of the visceral musculature observed in cross-section. (A–C) Virtual cross-sections of confocal Z-stacks of Platynereis larvae stained with DAPI and phalloidin. Dorsal side up. (A’–C’) Schematic drawings of the same individuals. Note the progressive formation of internal circular fibres around the gut, followed by the formation of external longitudinal fibres. Due to the complex three-dimensional organization of the somatic musculature, different subsets of somatic bundles are observed at different cross-sectional levels within a segment (compare with Figure 2B). The orientation of somatic myofibres is represented based on information from 3D reconstructions (Figure 2B) and TEM (Figure 2C–M). Note the progressive appearance of endodermal nuclei, indicating stepwise cellularization of the midgut from the macromeres. The outline of the endodermal epithelium could be visualized by enhancing the intensity of the green (phalloidin) channel and was drawn from that information in panels (A’–C’). gm: gut muscles, im: intrinsic muscles, vlm: ventral longitudinal muscles, ppm: parapodial muscles, vom: ventral oblique muscles, ach: axochord, dlm: dorsal longitudinal muscles, ch:…

Figure 3.. Expression of smooth and striated muscle markers in Platynereis larvae. Animals have been stained by WMISH and observed in bright-field Nomarski microscopy. Ventral views, anterior side up. Scale bar: 25 μm. (A–D) Expression patterns of the striated marker ST-MHC and the smooth marker SM-MHC. These expression patterns are representative of the entire striated and smooth effector module (see Figure 3—figure supplement 1 and Figure 3—figure supplement 3). Note that SM-MHC (panel B) is expressed around the forming midgut and hindgut (dotted white line) as well as in the stomodeal sheath (white arrows) and in lateral cells in the parapodia. The identity of these cells is unknown, but preliminary observations suggest they will become part of the nephridial tubule/nephridiopore complex that opens at the base of the parapodia in annelids. Asterisk: stomodeum. (E) Table summarizing the expression patterns of smooth and striated markers in Platynereis and vertebrate muscles. (*) indicates that Platynereis and vertebrate Calponin are mutually most resembling by domain structure, but not one-to-one orthologs, as independent duplications in both lineages have given rise to more broadly…

Figure 3—figure supplement 1.. Expression of striated muscle markers in Platynereis larvae. (A–O) Larvae stained by WMISH and observed in bright-field Nomarski microscopy. Ventral views, anterior side up. Scale bar is 20 μm for 48 hpf and 25 μm for the two other stages. (P) Foregut musculature visualized by rhodamine-phalloidin fluorescent staining. Z-projection of confocal planes. Ventral view, anterior side up. Scale bar: 20 μm. (Q) TEM micrograph of a cross-section of the foregut. Foregut muscles are colored green, axochord orange, ventral oblique muscles pink, ventral nerve cord yellow. Inset: zoom on the area in the red dashed box with enhanced contrast to visualize oblique striation. Scale bar: 10 μm. (R–W) WMISH for striated muscle markers expression in the foregut observed in Nomarski bright field microscopy, ventral views, anterior side up. Scale bar: 20 μm. DOI: http://dx.doi.org/10.7554/eLife.19607.007

Figure 3—figure supplement 2.. Expression of striated muscle markers in the six dpf Platynereis larva. Animals have been stained by WMISH and observed by confocal microscopy (DAPI fluorescence and NBT/BCIP 633 nm reflection). All striated effector genes are expressed in all somatic muscles examined. The transcription factor myoD is detectable in the axochord and in ventral longitudinal muscles, but not in other muscles. DOI: http://dx.doi.org/10.7554/eLife.19607.008

Figure 3—figure supplement 3.. Expression of smooth muscle markers in Platynereis larvae. (A–F) Animals are stained by WMISH and observed by Nomarski bright-field microscopy. Ventral views, anterior side up. Yellow arrows: expression in the foregut mesoderm. White dashed lines: outline of the midgut and hindgut (or their anlage at three dpf). Asterisk: stomodeum. (G) Schematic drawing of a six dpf larva (ventral view, anterior is up) representing gene expression in the forming tripartite gut (compare to Figure 2—figure supplement 1). (H–M) Molecular profile of the pulsatile dorsal vessel. All panels show 2-month-old juvenile worms. (H,I) Maximal Z-projections of confocal stacks. Dorsal view, anterior is up. (H) Dorsal musculature of a juvenile Platynereis dumerilii individual visualized by phalloidin-rhodamine (green) together with nuclear (DAPI, blue) and membrane (FM-464FX, red) stainings. The heart tube lies on the dorsal side, bordered by the somatic dorsal longitudinal muscles (dlm). (I) Expression of SM-MHC in the heart tube visualized by WMISH. (J) Virtual cross-section of the individual shown in A. Dorsal side up. Note the continuity of the muscular heart tube with gut musculature. dlm: dorsal…

Figure 3—figure supplement 4.. Molecular profile of midgut muscles in the six dpf larva. All panels are Z-projections of confocal planes, ventral views, anterior side up. Blue: DAPI, red: NBT/BCIP precipitate. White dashed line: midgut/hindgut, yellow dashed ellipse: stomodeum. (A–E) Smooth markers expression. White arrows indicate somatic expression of GATA456 in the ventral oblique muscles. (F–K) Striated markers expression; none of them is detected in any gut cell. White arrows: somatic expression. Scale bar: 20 μm in all panels. DOI: http://dx.doi.org/10.7554/eLife.19607.010

Figure 3—figure supplement 5.. General muscle markers are expressed in both smooth and striated muscles. All panels show gene expression visualized by WMISH. (A–F) actin expression. (A–B) bright-field micrographs in Nomarski optics. (A) is an apical view, (B) is a ventral view. Abbreviations: dlm, dorsal longitudinal muscles; vc, ventral mesodermal cells, likely representing future ventral musculature. (C–F) 3D rendering of confocal imaging of NBT/BCIP precipitate. (C,E) ventral views, anterior side up. (D,F) ventrolateral views, anterior side up. Abbreviations are as in Figure 2—figure supplement 2. (G–M) Z-projections of confocal stacks. Blue is DAPI, red is reflection signal of NBT/BCIP precipitate. (G–L) Ventral views, anterior side up. White dashed line: midgut, yellow ellipse: foregut. White arrows: somatic muscle expression. Abbreviations: f.m.: foregut muscles; r.a.: reflection artifact. (M) Expression in individual somatic muscles. Scale bar: 20 μm in all panels. DOI: http://dx.doi.org/10.7554/eLife.19607.011

Figure 4.. Contraction speed quantifications of smooth and striated muscles. (A–B) Snapshots of a time lapse live confocal imaging of a late nectochaete larva expressing fluorescent markers. Ventral view of the two posterior-most segments, anterior is up. (C) Snapshots of a time-lapse live confocal imaging of a three dpf larva expressing GCaMP6s. Dorsal view, anterior is up. (D–E) Two consecutive snapshots on the left dorsal longitudinal muscle of the larva shown in C, showing muscle contraction. (F) Quantification of smooth and striated muscle contraction speeds (see Experimental procedures and Figure 4—source data 1), p-value by Mann-Whitney’s U test. Each point represents a biological replicate (see Materials and methods). (G) Snapshot of a time-lapse live confocal imaging of a late nectochaete larva. Ventral view, anterior is up. Optical longitudinal section at the midgut level. DOI: http://dx.doi.org/10.7554/eLife.19607.012 10.7554/eLife.19607.013Figure 4—source data 1.Contraction speed values measured for somatic and visceral muscles.DOI: http://dx.doi.org/10.7554/eLife.19607.013

Figure 5.. Platynereis gut peristalsis is independent of nervous inputs and dependent on gap junctions. (A) Two-month-old juvenile mounted in 3% low-melting point (LMP) agarose for live imaging. (B) Snapshots of a confocal live time-lapse imaging of the animal shown in A. Gut is observed by detecting fluorescence of the vital membrane dye FM-464FX. (C) Kymograph of gut peristalsis along the line of interest in (B). Contraction waves appear as dark stripes. A series of consecutive contraction waves is called a contraction event: here, two contraction waves are visible, which make up one contraction event with a recurrence of 2. (D) Quantification of the propagation speed of peristaltic contraction waves in mock (DMSO)-treated individuals and Brefeldin-A-treated individuals (inhibiting neurotransmission). Speed is calculated from kymographs (see Materials and methods and Figure 5—source data 1), p-value by Mann-Whitney’s U test. Each point represents a contraction wave. Five biological replicates for each category (see Materials and methods). (E,F) Same as in E, but showing respectively the frequency of initiation and the recurrence of contraction events. Each point represents a biological…