1 The evolutionary origin of bilaterian smooth and striated

Transcription

bioRxiv preprint first posted online Jul. 20, 2016; doi: http://dx.doi.org/10.1101/064881. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
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The evolutionary origin of bilaterian smooth and striated myocytes
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Thibaut Brunet1, Antje H. L. Fischer1,2, Patrick R. H. Steinmetz1,3, Antonella Lauri1,4,
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Paola Bertucci1, and Detlev Arendt1,*
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Meyerhofstraße, 1, 69117 Heidelberg, Germany
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Present address: Ludwig-Maximilians University Munich, Biochemistry, FeodorLynen Straße 17, 81377 Munich
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Developmental Biology Unit, European Molecular Biology Laboratory,
Present address: Sars International Centre for Marine Molecular Biology,
University of Bergen, Thormøhlensgate 55, 5008 Bergen, Norway.
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Present address: Institute for Biological and Medical Imaging (IBMI), Helmholtz
Zentrum München, Neuherberg, Germany
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* For correspondence: [email protected]
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Abstract
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The dichotomy between smooth and striated myocytes is fundamental for bilaterian
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musculature, but its evolutionary origin remains unsolved. Given their absence in
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Drosophila and Caenorhabditis, smooth muscles have so far been considered a
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vertebrate innovation. Here, we characterize expression profile, ultrastructure,
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contractility and innervation of the musculature in the marine annelid Platynereis
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dumerilii and identify smooth muscles around the midgut, hindgut and heart that
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resemble their vertebrate counterparts in molecular fingerprint, contraction speed,
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and nervous control. Our data suggest that both visceral smooth and somatic
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striated myocytes were present in the protostome-deuterostome ancestor, and that
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smooth myocytes later co-opted the striated contractile module repeatedly – for
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example in vertebrate heart evolution. During these smooth-to-striated myocyte
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conversions the core regulatory complex of transcription factors conveying myocyte
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identity remained unchanged, reflecting a general principle in cell type evolution.
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Introduction
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Musculature is composed of myocytes that are specialized in active contraction (Schmidt-
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Rhaesa 2007). This function relies on actomyosin, a contractile module that dates back to
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stem eukaryotes (Brunet & Arendt 2016) and incorporated accessory proteins of pre-
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metazoan origin (Steinmetz et al. 2012). Two fundamentally distinct types of myocytes
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are distinguished based on ultrastructural appearance. In striated myocytes, actomyosin
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myofibrils are organized in aligned repeated units (sarcomeres) separated by transverse
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‘Z discs’, while in smooth myocytes adjacent myofibrils show no clear alignment and are
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separated by scattered “dense bodies” (Figure 1A). In vertebrates, striated myocytes are
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found in voluntary skeletal muscles, but also at the anterior and posterior extremities of
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the digestive tract (anterior esophagus muscles and external anal sphincter), and in the
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muscular layer of the heart; smooth myocytes are found in involuntary visceral
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musculature that ensures slow, long-range deformation of internal organs. This includes
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the posterior esophagus and the rest of the gut, but also blood vessels, and most of the
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urogenital system. In stark contrast, in the fruit fly Drosophila virtually all muscles are
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striated, including gut visceral muscles (Anderson & Ellis 1967; Goldstein & Burdette
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1971; Paniagua et al. 1996); the only exception are poorly-characterized multinucleated
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smooth muscles around the testes (Susic-Jung et al. 2012). Also, in the nematode
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Caenorhabditis, somatic muscles are striated, while the short intestine and rectum
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visceral myocytes are only one sarcomere-long and thus hard to classify (White 1988;
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Corsi et al. 2000).
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The evolutionary origin of smooth versus striated myocytes in bilaterians accordingly
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remains unsolved. Ultrastructural studies have consistently documented the presence of
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striated somatic myocytes in virtually every bilaterian group (Schmidt-Rhaesa 2007) and
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in line with this, the comparison of Z-disc proteins supports homology of striated
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myocytes across bilaterians (Steinmetz et al. 2012). The origin of smooth myocyte types
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however is less clear. Given the absence of smooth muscles from fly and nematode, it has
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been proposed that visceral smooth myocytes represent a vertebrate novelty, which
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evolved independently from non-muscle cells in the vertebrate stem line (Goodson &
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Spudich 1993; OOta & Saitou 1999). However, smooth muscles are present in many
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other bilaterian groups, suggesting instead their possible presence in urbilaterians and
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secondary loss in arthropods and nematodes. Complicating the matter further,
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intermediate ultrastructures between smooth and striated myocytes have been reported,
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suggesting interconversions (reviewed in (Schmidt-Rhaesa 2007))
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Besides ultrastructure, the comparative molecular characterization of cell types can be
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used to build cell type trees (Arendt 2003; Arendt 2008; Wagner 2014; Musser & Wagner
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2015). Cell type identity is established via the activity of transcription factors acting as
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terminal selectors (Hobert 2016) and forming “core regulatory complexes” (CRCs;
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(Arendt et al. 2016; Wagner 2014)), which directly activate downstream effector genes.
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This is exemplified for vertebrate myocytes in Figure 1B. In all vertebrate myocytes,
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transcription factors of the Myocardin family (MASTR in skeletal muscles, Myocardin in
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smooth and cardiac muscles) directly activate effector genes encoding contractility
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proteins (Fig. 1B) (Creemers et al. 2006; Meadows et al. 2008; Wang & Olson 2004;
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Wang et al. 2003)). They heterodimerize with MADS-domain factors of the Myocyte
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Enhancer Factor-2 (Mef2) (Black & Olson 1998; Blais et al. 2005; Molkentin et al. 1995;
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Wales et al. 2014) and Serum Response Factor (SRF) families (Carson et al. 1996;
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Nishida et al. 2002). Other myogenic transcription factors are specific for different types
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of striated and smooth myocytes. Myogenic Regulatory Factors (MRF) family members,
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including MyoD and its paralogs Myf5, Myogenin, and Mrf4/Myf6 (Shi & Garry 2006),
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directly control contractility effector genes in skeletal (and esophageal) striated
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myocytes, cooperatively with Mef2 (Blais et al. 2005; Molkentin et al. 1995) – but are
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absent from smooth and cardiac muscles. In smooth and cardiac myocytes, this function
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is ensured by NK transcription factors (Nkx3.2/Bapx and Nkx2.5/Tinman, respectively),
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GATA4/5/6, and Fox transcription factors (FoxF1 and FoxC1, respectively), which bind
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to SRF and Mef2 to form CRCs directly activating contractility effector genes (Durocher
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et al. 1997; Hoggatt et al. 2013; Lee et al. 1998; Morin et al. 2000; Nishida et al. 2002;
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Phiel et al. 2001) (Figure 1B).
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Regarding effector proteins (Figure 1B), (Kierszenbaum & Tres 2015), all myocytes
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express distinct isoforms of the myosin heavy chain: the striated myosin heavy chain ST-
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MHC (which duplicated into cardiac, fast skeletal, and slow skeletal isoforms in
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vertebrates) and the smooth/non-muscle myosin heavy chain SM-MHC (which duplicated
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in vertebrates into smooth myh10, myh11 and myh14, and non-muscle myh9) (Steinmetz
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et al. 2012). The different contraction speeds of smooth and striated muscles are due to
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the distinct kinetic properties of these molecular motors (Bárány 1967). In both myocyte
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types, contraction occurs in response to calcium, but the responsive proteins differ
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(Alberts et al. 2014): the Troponin complex (composed of Troponin C, Troponin T and
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Troponin I) for striated muscles, Calponin and Caldesmon for smooth muscles. In both
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myocyte types, calcium also activates the Calmodulin/Myosin Light Chain Kinase
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pathway (Kamm & Stull 1985; Sweeney et al. 1993). Striation itself is implemented by
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specific effectors, including the long elastic protein Titin (Labeit & Kolmerer 1995)
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(which spans the entire sarcomere and confers it elasticity and resistance) and
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ZASP/LBD3 (Z-band Alternatively Spliced PDZ Motif/LIM-Binding Domain 3), which
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binds actin and stabilizes sarcomeres during contraction (Au et al. 2004; Zhou et al.
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2001). The molecular study of Drosophila and Caenorhabditis striated myocytes
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revealed important commonalities with their vertebrate counterparts, including the
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Troponin complex (Beall & Fyrberg 1991; Fyrberg et al. 1994; Fyrberg et al. 1990;
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Marín et al. 2004; Myers et al. 1996), and a conserved role for Titin (Zhang et al. 2000)
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and ZASP/LBD3 (Katzemich et al. 2011; McKeown et al. 2006) in the striated
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architecture.
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Finally, smooth and striated myocytes also differ physiologically. All known striated
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myocyte types (apart from the myocardium) strictly depend on nervous stimulations for
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contraction, exerted by innervating motor neurons. In contrast, gut smooth myocytes are
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able to generate and propagate automatic (or “myogenic”) contraction waves responsible
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for digestive peristalsis in the absence of nervous inputs (Faussone‐Pellegrini &
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Thuneberg 1999; Sanders et al. 2006). These autonomous contraction waves are
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modulated by the autonomic nervous system (Silverthorn 2015). Regarding overall
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contraction speed, striated myocytes have been measured to contract 10 to 100 times
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faster than their smooth counterparts (Bárány 1967).
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To elucidate the evolutionary origin and diversification of bilaterian smooth and striated
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myocytes,
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characterization of the myocyte complement in the marine annelid Platynereis dumerilii,
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which belongs to the Lophotrochozoa. Strikingly, as of now, no invertebrate smooth
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visceral muscle has been investigated on a molecular level (Hooper & Thuma 2005;
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Hooper et al. 2008). Platynereis has retained more ancestral features than flies or
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nematodes and is thus especially suited for long-range comparisons (Raible et al. 2005;
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Denes et al. 2007). Also, other annelids such as earthworms have been reported to
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possess both striated somatic and midgut smooth visceral myocytes based on electron
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microscopy (Anderson & Ellis 1967). Our study reveals the parallel presence of smooth
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myocytes in the musculature of midgut, hindgut, and pulsatile dorsal vessel and of
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striated myocytes in the somatic musculature and the foregut. Platynereis smooth and
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striated myocytes closely parallel their vertebrate counterparts in ultrastructure, molecular
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profile, contraction speed, and reliance on nervous inputs, thus supporting the ancient
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existence of a smooth-striated duality in protostome/deuterostome ancestors.
we
provide
an
in-depth
ultrastructural,
molecular
and
functional
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Results
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Platynereis midgut and hindgut muscles are smooth, while foregut and somatic muscles
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are striated
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Differentiation of the Platynereis somatic musculature has been documented in much
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detail (Fischer et al. 2010) and, in five days old young worms, consists of ventral and
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dorsal longitudinal muscles, oblique and parapodial muscles, head muscles and the
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axochord (Lauri et al. 2014). At this stage, the first Platynereis visceral myocytes become
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detectable around the developing tripartite gut, which is subdivided into foregut, midgut
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and hindgut (based on the conserved regional expression of foxA, brachyury and hnf4 gut
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specification factors (Martín-Durán & Hejnol 2015); Figure 2-supplement 1). At 7 days
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post fertilization (dpf), visceral myocytes form circular myofibres around the foregut, and
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scattered longitudinal and circular fibres around midgut and hindgut (Figure 2A), which
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expand by continuous addition of circular and longitudinal fibres to completely cover the
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dorsal midgut at 11dpf (Figure 2A) and finally form a continuous muscular orthogon
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around the entire midgut and hindgut in the 1.5 months-old juvenile (Figure 2A).
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We then proceeded to characterize the ultrastructure of Platynereis visceral and somatic
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musculature by transmission electron microscopy (Figure 2C-M). All somatic muscles
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and anterior foregut muscles display prominent oblique striation with discontinuous Z-
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elements (Figure 2C-H; compare Figure 1A), as typical for protostomes (Burr & Gans
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1998; Mill & Knapp 1970; Rosenbluth 1972). To the contrary, visceral muscles of the
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posterior foregut, midgut and hindgut are smooth with scattered dense bodies (Figure 2I-
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M). The visceral muscular orthogon is partitioned into an external longitudinal layer and
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an internal circular layer (Figure 2J), as in vertebrates (Marieb & Hoehn 2015) and
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arthropods (Lee et al. 2006). Thus, according to ultrastructural appearance, Platynereis
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has both somatic (and anterior foregut) striated muscles and visceral smooth muscles.
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The molecular profile of smooth and striated myocytes
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We then set out to molecularly characterize annelid smooth and striated myocytes via a
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candidate gene approach. As a starting point, we investigated, in the Platynereis genome,
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the presence of regulatory and effector genes specific for smooth and/or striated
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1
myocytes in the vertebrates. We found striated muscle-specific and smooth muscle/non-
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muscle isoforms of both myosin heavy chain (consistently with published phylogenies
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(Steinmetz et al. 2012)) and myosin regulatory light chain. We also identified homologs
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of genes encoding calcium transducers (calponin for smooth muscles; troponin I and
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troponin T for striated muscles), striation structural proteins (zasp/lbd3 and titin), and
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terminal selectors for the smooth (nk3, foxF, and gata456) and striated phenotypes
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(myoD).
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We investigated expression of these markers by whole-mount in situ hybridization
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(WMISH). Striated effectors are expressed in both somatic and foregut musculature
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(Figure 3A,C; Figure 3—supplement 1). Expression of all striated effectors was observed
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in every somatic myocyte group by confocal imaging with cellular resolution (Figure 3—
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supplement 2). Interestingly, myoD is exclusively expressed in longitudinal striated
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muscles, but not in other muscle groups (Figure 3—supplement 2).
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The expression of smooth markers is first detectable at 3 dpf in a small triangle-shaped
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group of mesodermal cells posteriorly abutting the macromeres (which will form the
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future gut) (Figure 3B, Figure 3—supplement 3A-D,H). Double WMISH shows that
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these cells coexpress smooth markers and the muscle marker actin (Figure 3—
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supplement 3I-K), supporting the idea that they are forming gut myocytes. At this stage,
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smooth markers are also expressed in the foregut mesoderm (Figure 3B, Figure 3—
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supplement 3A-D,H, yellow arrows). At 6 dpf, expression of all smooth markers (apart
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from nk3) is maintained in the midgut and hindgut differentiating myocytes (Figure 3D,
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Figure 3—supplement 3E-G, Figure 3—supplement 4A-E) but smooth effectors
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disappear from the foregut, which turns on striated markers instead (Figure 3—
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supplement 1R-W) – reminiscent of the replacement of smooth fibres by striated fibres
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during development of the vertebrate anterior esophageal muscles (Gopalakrishnan et al.
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2015). Finally, in 2 months-old juvenile worms, smooth markers are also detected in the
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dorsal pulsatile vessel (Figure 3—supplement 3L-Q) – considered equivalent to the
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vertebrate heart (Saudemont et al. 2008) but, importantly, of smooth ultrastructure in
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polychaetes (Spies 1973; Jensen 1974). None of the striated markers is expressed around
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the midgut or the hindgut (Figure 3—supplement 4F-K), or in the dorsal vessel (Figure
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3—supplement 3P). Taken together, these results strongly support conservation of the
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molecular fingerprint of both smooth and striated myocytes between annelids and
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vertebrates.
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We finally investigated general muscle markers that are shared between smooth and
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striated muscles. These include actin, mef2 and myocardin – which duplicated into
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muscle type-specific paralogs in vertebrates, but are still present as single-copy genes in
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Platynereis. We found them to be expressed in the forming musculature throughout larval
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development (Figure 3—supplement 5A-F), and confocal imaging at 6 dpf confirmed
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expression of all 3 markers in both visceral (Figure 3—supplement 5G-L) and somatic
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muscles (Figure 3—supplement 5M).
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Smooth and striated muscles differ in contraction speed
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We then characterized the contraction speed of the two myocyte types in Platynereis by
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measuring myofibre length before and after contraction. Live confocal imaging of
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contractions in Platynereis larvae with fluorescently labeled musculature (Movie 1,
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Movie 2) gave a striated contraction rate of 0.55±0.27 s-1 (Figure 4A-E) and a smooth
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myocyte contraction rate of 0.07±0.05 s-1 (Figure 4G). As in vertebrates, annelid striated
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myocytes thus contract nearly one order of magnitude faster than smooth myocytes
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(Figure 4F).
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Striated, but not smooth, muscle contraction depends on nervous inputs
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Finally, we investigated the nervous control of contraction of both types of muscle cells.
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In vertebrates, somatic muscle contraction is strictly dependent on neuronal inputs. By
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contrast, gut peristalsis is automatic (or myogenic – i.e., does not require nervous inputs)
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in vertebrates, cockroaches (Nagai & Brown 1969), squids (Wood 1969), snails (Roach
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1968), holothurians, and sea urchins (Prosser et al. 1965). The only exceptions appear to
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be bivalves and malacostracans (crabs, lobster and crayfish), in which gut motility is
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neurogenic (Prosser et al. 1965). Regardless of the existence of an automatic component,
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the gut is usually innervated by nervous fibres modulating peristalsis movements (Wood
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1969; Wu 1939).
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Gut peristalsis takes place in Platynereis larvae and juveniles from 6 dpf onwards (Movie
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3), and we set out to test whether nervous inputs were necessary for it to take place. We
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treated 2 months-old juveniles with 180 µM Brefeldin A, an inhibitor of vesicular traffic
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which prevents polarized secretion (Misumi et al. 1986) and neurotransmission (Malo et
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al. 2000). Treatment stopped locomotion in all treated individuals, confirming that
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neurotransmitter release by motor neurons is required for somatic muscles contraction,
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while DMSO-treated controls were unaffected. On the other hand, vigorous gut
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peristalsis movements were maintained in Brefeldin A-treated animals (Movie 4).
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Quantification of the propagation speed of the peristalsis wave (Figure 5A-D; see
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Material and Methods) indicated that contractions propagated significantly faster in
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Brefeldin A-treated individuals than in controls. The frequency of wave initiation and
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their recurrence (the number of repeated contraction waves occurring in one
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uninterrupted sequence) did not differ significantly in Brefeldin A-treated animals
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(Figure 5E,F). These results indicate that, as in vertebrates, visceral smooth muscle
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contraction and gut peristalsis do not require nervous (or secretory) inputs in Platynereis.
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An enteric nervous system is present in Platynereis
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In vertebrates, peristaltic contraction waves are initiated by self-excitable myocytes
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(Interstitial Cajal Cells) and propagate across other smooth muscles by gap junctions
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ensuring direct electrical coupling (Faussone‐Pellegrini & Thuneberg 1999; Sanders et
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al. 2006). We tested the role of gap junctions in Platynereis gut peristalsis by treating
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animals with 2.5 mM 2-octanol, which inhibits gap junction function in both insects
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(Bohrmann & Haas-Assenbaum 1993; Gho 1994) and vertebrates (Finkbeiner 1992). 2-
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octanol abolishes gut peristalsis, both in the absence and in the presence of Brefeldin A
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(Figure 5G), indicating that propagation of the peristalsis wave relies on direct coupling
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between smooth myocytes via gap junctions.
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The acceleration of peristalsis upon Brefeldin A treatment suggests that secretory (and
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possibly neural) inputs modulate gut contractions (with a net inhibitory effect). We thus
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investigated the innervation of the Platynereis gut. Immunostainings of juvenile worms
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for acetylated tubulin revealed a dense, near-orthogonal nerve net around the entire gut
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(Figure 6A), which is tightly apposed to the visceral muscle layer (Figure 6C) and
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includes serotonergic neurites (Figure 6B,C) and cell bodies (Figure 6D). Interestingly,
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some enteric serotonergic cell bodies are devoid of neurites, thus resembling the
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vertebrate (non-neuronal) enterochromaffine cells – endocrine serotonergic cells residing
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around the gut and activating gut peristalsis by direct serotonin secretion upon
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mechanical stretch (Bulbring & Crema 1959).
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Discussion
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Smooth and striated myocyte coexisted in bilaterian ancestors
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Our study represents the first molecular characterization of protostome visceral smooth
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musculature (Hooper & Thuma 2005; Hooper et al. 2008). The conservation of molecular
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signatures for both smooth and striated myocytes indicates that a dual musculature
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already existed in bilaterian ancestors: a fast striated somatic musculature (possibly also
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present around the foregut – as in Platynereis, vertebrates (Gopalakrishnan et al. 2015)
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and sea urchins (Andrikou et al. 2013; Burke 1981)), under strict nervous control; and a
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slow smooth visceral musculature around the midgut and hindgut, able to undergo
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automatic peristalsis thanks to self-excitable myocytes directly coupled by gap junctions,
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and undergoing modulation by nervous and paracrine inputs. In striated myocytes, a core
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regulatory complex (CRC) involving Mef2 and Myocardin directly activated striated
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contractile effector genes such as ST-MHC, ST-MRLC and the Troponin genes (Figure
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7—supplement 1). Notably, myoD might have been part of the CRC in only part of the
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striated myocytes, as it is only detected in longitudinal muscles in Platynereis. The
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absence of myoD expression in other annelid muscle groups is in line with the “chordate
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bottleneck” concept (Thor & Thomas 2002), according to which specialization for
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undulatory swimming during early chordate evolution would have fostered exclusive
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reliance on trunk longitudinal muscles, and loss of other muscle types. In smooth
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myocytes, a CRC composed of NK3, FoxF and GATA4/5/6 together with Mef2 and
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Myocardin activated the smooth contractile effectors SM-MHC, SM-MRLC and calponin
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(Figure 7—supplement 1). In spite of their absence in flies and nematodes, gut myocytes
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of smooth ultrastructure are widespread in other bilaterians, and an ancestral state
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reconstruction retrieves them as present in the last common protostome/deuterostome
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ancestor with high confidence (Figure 7—supplement 2), supporting our homology
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hypothesis.
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immunoreactivity in intestinal muscles of earthworms (Royuela et al. 1997) and snails
8
(which also lack immunoreactivity for Troponin T) (Royuela et al. 2000).
Our
results
are
consistent
with
previous
reports
of
Calponin
9
10
Origin of smooth and striated myocytes by cell type individuation
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How did smooth and striated myocytes diverge in evolution? Figure 7 presents a
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comprehensive cell type tree for the evolution of myocytes, with a focus on Bilateria.
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This tree illustrates the divergence of the two muscle cell types by progressive
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partitioning of genetic information in evolution – a process called individuation (Wagner
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2014; Arendt et al. 2016). The individuation of fast and slow contractile cells involved
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two complementary processes: (1) changes in CRC (black circles, Figure 7) and (2)
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emergence of novel genes encoding new cellular modules, or apomeres (Arendt et al.
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2016) (grey squares, Figure 7).
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Around a common core formed by the Myocardin:Mef2 complex (both representing
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transcription factors of pre-metazoan ancestry (Steinmetz et al. 2012)), smooth and
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striated CRCs incorporated different transcription factors implementing the expression of
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distinct effectors (Figure 1B; Figure 7—supplement 1) – notably the bilaterian-specific
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1
bHLH factor MyoD (Steinmetz et al. 2012) and GATA4/5/6, which arose by bilaterian-
2
specific duplication of a single ancient pan-endomesodermal GATA transcription factor
3
(Martindale et al. 2004; Leininger et al. 2014).
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Regarding the evolution of myocyte-specific apomeres, one prominent mechanism of
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divergence has been gene duplications. While the MHC duplication predated metazoans,
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other smooth and striated-specific paralogs only diverged in bilaterians. Smooth and
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striated MRLC most likely arose by gene duplication in the bilaterian stem-line
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(Supplementary Figure 1). Myosin essential light chain, actin and myocardin paralogs
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split even later, in the vertebrate stem-line (Figure 7). Similarly, smooth and non-muscle
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mhc and mrlc paralogs only diverged in vertebrates. The calponin-encoding gene
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underwent parallel duplication and subfunctionalization in both annelids and chordates,
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giving rise to both specialized smooth muscle paralogs and more broadly expressed
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copies with a different domain structure (Figure 7—supplement 3). This slow and
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stepwise nature of the individuation process is consistent with studies showing that
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recently evolved paralogs can acquire differential expression between tissues that
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diverged long before in evolution (Force et al. 1999; Lan & Pritchard 2016).
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Complementing gene duplication, the evolution and selective expression of entirely new
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apomeres also supported individuation: for example, Titin and all components of the
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Troponin complex are bilaterian novelties (Steinmetz et al. 2012). In vertebrates, the new
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gene caldesmon was incorporated in the smooth contractile module (Steinmetz et al.
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2012).
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1
Smooth to striated myocyte conversion
2
Strikingly, visceral smooth myocytes were previously assumed to be a vertebrate
3
innovation, as they are absent in fruit flies and nematodes (two groups which are in fact
4
exceptions in this respect, at least from ultrastructural criteria (Figure 7—supplement
5
2A)). This view received apparent support from the fact that the vertebrate smooth and
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non-muscle myosin heavy chains (MHC) arose by vertebrate-specific duplication of a
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unique ancestral bilaterian gene, orthologous to Drosophila non-muscle MHC (Goodson
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& Spudich 1993) – which our results suggest reflects instead gradual individuation of
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pre-existing cell types (see above). Strikingly, the striated gut muscles of Drosophila
10
closely resemble vertebrate and annelid smooth gut muscles by transcription factors
11
(nk3/bagpipe (Azpiazu & Frasch 1993), foxF/biniou (Jakobsen et al. 2007a; Zaffran et al.
12
2001)), even though they express the fast/striated contractility module (Fyrberg et al.
13
1994; Fyrberg et al. 1990; Marín et al. 2004). If smooth gut muscles are ancestral for
14
protostomes, as our results indicate, this suggests that the smooth contractile module was
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replaced by the fast/striated module in visceral myocytes during insect evolution.
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Interestingly, chromatin immunoprecipitation assays (Jakobsen et al. 2007a) show that
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the conserved visceral transcription factors foxF/biniou and nk3/bagpipe do not directly
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control contractility genes in Drosophila gut muscles (which are downstream mef2
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instead), but establish the morphogenesis and innervation of the visceral muscles, and
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control non-contractile effectors such as gap junctions – which are the properties these
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muscles seem to have conserved from their smooth ancestors. The striated gut myocytes
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of insects would thus represent a case of co-option of an effector module from another
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1
cell type, which happened at an unknown time during ecdysozoan evolution (Figure 7;
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Figure 7—supplement 1).
3
Another likely example of co-option is the vertebrate heart: vertebrate cardiomyocytes
4
are striated and express fast myosin and troponin, but resemble smooth myocytes by
5
developmental origin (from the splanchnopleura), function (automatic contraction and
6
coupling by gap junctions) and terminal selector profile (Figure 1B). These similarities
7
suggest that cardiomyocytes might stem from smooth myocytes that likewise co-opted
8
the fast/striation module. Indicative of this possible ancestral state, the Platynereis dorsal
9
pulsatile vessel (considered homologous to the vertebrate heart based on comparative
10
anatomy and shared expression of NK4/tinman (Saudemont et al. 2008)) expresses the
11
smooth, but not the striated, myosin heavy chain (Figure 3—supplement 3O-Q). An
12
ancestral state reconstruction based on ultrastructural data further supports the notion that
13
heart myocytes were smooth in the last common protostome/deuterostome ancestor, and
14
independently acquired striation in at least 5 descendant lineages (Figure 7—supplement
15
2B) – usually in species with large body size and/or fast metabolism.
16
17
Striated to smooth conversions
18
Smooth somatic muscles are occasionally found in bilaterians with slow or sessile
19
lifestyles – for example in the snail foot (Faccioni-Heuser et al. 1999; Rogers 1969), the
20
ascidian siphon (Meedel & Hastings 1993), and the sea cucumber body wall (Kawaguti &
21
Ikemoto 1965). As an extreme (and isolated) example, flatworms lost striated muscles
22
altogether, and their body wall musculature is entirely smooth (Rieger et al. 1991).
17
1
Interestingly, in all cases that have been molecularly characterized, smooth somatic
2
muscles express the same fast contractility module as their striated counterparts,
3
including ST-MHC and the Troponin complex – in ascidians (Endo & Obinata 1981;
4
Obinata et al. 1983), flatworms (Kobayashi et al. 1998; Witchley et al. 2013; Sulbarán et
5
al. 2015), and the smooth myofibres of the bivalve catch muscle (Nyitray et al. 1994;
6
Ojima & Nishita 1986). (It is unknown whether these also express zasp and titin in spite
7
of the lack of striation). This suggests that these are somatic muscles having secondarily
8
lost striation (in line with the sessile lifestyle of ascidians and bivalves, and with the
9
complete loss of striated muscles in flatworms). Alternatively, they might represent
10
remnants of ancestral smooth somatic fibres that would have coexisted alongside striated
11
somatic fibres in the last common protostome/deuterostome ancestor. Interestingly, the
12
fast contractile module is also expressed in acoel body wall smooth muscles (Chiodin et
13
al. 2011); since acoels belong to a clade that might have branched off before all other
14
bilaterians (Cannon et al. 2016) (but see (Telford & Copley 2016)), these could represent
15
fast-contracting myocytes that never evolved striation in the first place, similar to those
16
found in cnidarians. In all cases, the fast contractility module appears to represent a
17
consistent synexpression group (i.e. its components are reliably expressed together), and
18
a stable molecular profile of all bilaterian somatic muscles, regardless of the presence of
19
morphologically overt striation. This confirms the notion that, even in cases of
20
ambiguous morphology or ultrastructure, the molecular fingerprint of cell types holds
21
clue to their evolutionary affinities.
18
1
Implications for cell type evolution
2
In the above, genetically well-documented cases of cell type conversion (smooth to
3
striated conversion in insect visceral myocytes and vertebrate cardiomyocytes), cells kept
4
their ancestral CRC of terminal selector transcription factors, while changing the
5
downstream effector modules. This supports the recent notion that CRCs confer an
6
abstract identity to cell types, which remains stable in spite of turnover in downstream
7
effectors (Wagner 2014) – just as hox genes impart conserved abstract identity to
8
segments of vastly diverging morphologies (Deutsch 2005). Tracking cell type-specific
9
CRCs through animal phylogeny thus represents a powerful means to decipher the
10
evolution of cell types.
11
Pre-bilaterian origins
12
If the existence of fast-contracting striated and slow-contracting smooth myocytes
13
predated bilaterians – when and how did these cell types first split in evolution? The first
14
evolutionary event that paved the way for the diversification of the smooth and striated
15
contractility modules was the duplication of the striated myosin heavy chain-encoding
16
gene into the striated isoform ST-MHC and the smooth/non-muscle isoform SM-MHC.
17
This duplication occurred in single-celled ancestors of animals, before the divergence of
18
filastereans and choanoflagellates (Steinmetz et al. 2012). Consistently, both sm-mhc and
19
st-mhc are present in the genome of the filasterean Ministeria (though st-mhc was lost in
20
other single-celled holozoans) (Sebé-Pedrós et al. 2014). Interestingly, st-mhc and sm-
21
mhc expression appears to be segregated into distinct cell types in sponges, cnidarians
22
(Steinmetz et al. 2012), and ctenophores (Dayraud et al. 2012), suggesting that a cell type
19
1
split between slow and fast contractile cells is a common feature across early-branching
2
metazoans (Figure 7). Given the possibility of MHC isoform co-option (as outlined
3
above), it is yet unclear whether this split happened once or several times. The affinities
4
of bilaterians and non-bilaterians contractile cells remain to be tested from data on the
5
CRCs establishing contractile cell types in non-bilaterians.
6
7
Conclusions
8
Our results indicate that the split between visceral smooth myocytes and somatic striated
9
myocytes is the result of a long individuation process, initiated before the last common
10
protostome/deuterostome ancestor. Fast- and slow-contracting cells expressing distinct
11
variants of myosin II heavy chain (ST-MHC versus SM-MHC) acquired increasingly
12
contrasted molecular profiles in a gradual fashion – and this divergence process continues
13
to this day in individual bilaterian phyla. Blurring this picture of divergence, co-option
14
events have led to the replacement of the slow contractile module by the fast one, leading
15
to smooth-to-striated myocyte conversions. Our study showcases the power of molecular
16
fingerprint comparisons centering on effector and selector genes to reconstruct cell type
17
evolution (Arendt 2008). In the bifurcating phylogenetic tree of animal cell types (Liang
18
et al. 2015), it remains an open question how the two types of contractile cells relate to
19
other cell types, such as neurons (Mackie 1970) or cartilage (Lauri et al. 2014).
20
21
22
20
1
Material and Methods
2
3
Immunostainings and in situ hybridizations
4
Immunostaining, rhodamine-phalloidin staining, and simple and double WMISH were
5
performed according to previously published protocols (Lauri et al. 2014). For all
6
stainings not involving phalloidin, the following modifications: animals were mounted in
7
97% TDE/3% PTw for imaging following (Asadulina et al. 2012). Phalloidin-stained
8
larvae were mounted in 1% DABCO/glycerol instead, as TDE was found to quickly
9
disrupt phalloidin binding to F-actin. Confocal imaging of stained larvae was performed
10
using a Leica SPE and a Leica SP8 microscope. Stacks were visualized and processed
11
with ImageJ 1.49v. 3D renderings were performed with Imaris 8.1. Bright field Nomarski
12
microscopy was performed on a Zeiss M1 microscope. Z-projections of Nomarski stacks
13
were performed using Helicon Focus 6.7.1. 14
15
Transmission electron microscopy
16
TEM was performed as previously published (Lauri et al. 2014).
17
18
Pharmacological treatments
19
Brefeldin A was purchased from Sigma Aldrich (B7561) and dissolved in DMSO to a
20
final concentration of 5 mg/mL. Animals were treated with 50 µg/mL Brefeldin A in 6-
21
well plates filled with 5 mL filtered natural sea water (FNSW). Controls were treated
22
with 1% DMSO (which is compatible with Platynereis development and survival without
23
noticeable effect). Other neurotransmission inhibitors had no effect on Platynereis (as
21
1
they elicited no impairment of locomotion): tetanus toxic (Sigma Aldrich T3194; 100
2
µg/mL stock in distilled water) up to 5 µg/mL; TTX (Latoxan, L8503; 1 mM stock) up to
3
10 µM; Myobloc (rimabotulinum toxin B; Solstice Neurosciences) up to 1%; saxitoxin 2
4
HCl (Sigma Aldrich NRCCRMSTXF) up to 1%; and neosaxitoxin HCl (Sigma Aldrich
5
NRCCRMNEOC) up to 1%. (±)-2-Octanol was purchased from Sigma Aldrich and
6
diluted to a final concentration of 2.5 mM (2 µL in 5 mL FNSW). (±)-2-Octanol
7
treatment inhibited both locomotion and gut peristalsis, in line with the importance of gap
8
junctions in motor neural circuits (Kawano et al. 2011; Kiehn & Tresch 2002).
9
10
11
Live imaging of contractions
12
13
Animals were mounted in 3% low melting point agarose in FNSW (2-
14
Hydroxyethylagarose, Sigma Aldrich A9414) between a slide and a cover slip (using 5
15
layers of adhesive tape for spacing) and imaged with a Leica SP8 confocal microscope.
16
Fluorescent labeling of musculature was achieved either by microinjection of mRNAs
17
encoding GCaMP6s, LifeAct-EGFP or H2B-RFP, or by incubation in 3 µM 0.1% FM-
18
464FX (ThermoFisher Scientific, F34653). Contraction speed was calculated as (l2-
19
l1)/(l1*t), where l1 is the initial length, l2 the length after contraction, and t the duration
20
of the contraction. Kymographs and wave speed quantifications were performed with the
21
ImageJ Kymograph plugin: http://www.embl.de/eamnet/html/kymograph.html
22
23
22
1
Ancestral state reconstruction
2
Ancestral state reconstructions were performed with Mesquite 3.04 using the Maximum
3
Likelihood and Parsimony methods.
4
5
Cloning
6
The following primers were used for cloning Platynereis genes using a mixed stages
7
Platynereis cDNA library (obtained from 1, 2, 3, 5, 6, 10, and 14-days old larvae) and
8
either the HotStart Taq Polymerase from Qiagen or the Phusion polymerase from New
9
England BioLabs (for GC-rich primers):
10
Gene name
Forward primer
Reverse primer
foxF
CCCAGTGTCTGCATCCTTGT
CATGGGCATTGAAGGGGAGT
zasp
CATACCAGCCATCCCGTCC
AAATCAGCGAACTCCAGCGT
troponin T
TTCTGCAGGGCGCAAAGTCA
CGCTGCTGTTCCTTGAAGCG
SM-MRLC
TGGTGTTTGCAGGGCGGTCA
GGTCCATACCGTTACGGAAGCTTTT
calponin
ACGTGCGGTTTACGATTGGA
GCTGGCTCCTTGGTTTGTTC
transgelin1
GCTGCCAAGGGAGCTGACGC ACAAAGAGCTTGTACCACCTCACCC
myocardin
GACACCAGTCCGAAGCTTGA
CGTGGTAGTAGTCGTGGTCG
11
12
13
The following genes were retrieved from an EST plasmid stock: SM-MHC (as two
14
independent clones that gave identical expression patterns) and ST-MRLC.
23
1
Other genes were previously published: NK3 (Saudemont et al. 2008), actin and ST-MHC
2
(under the name mhc1-4) (Lauri et al. 2014) and GATA456 (Gillis et al. 2007).
3
Acknowledgements
4
5
We thank Kaia Achim for the hnf4 plasmid, Pedro Machado (Electron Microscopy Core
6
Facility, EMBL Heidelberg) for embedding and sectioning samples for TEM, and the
7
Arendt lab for feedback on the project. The work was supported by the European
8
Research Council “Brain Evo-Devo” grant (TB, PB and DA), European Union’s Seventh
9
Framework Programme project “Evolution of gene regulatory networks in animal
10
development (EVONET)” [215781-2] (AL), the Zoonet EU-Marie Curie early training
11
network [005624] (AF), the European Molecular Biology Laboratory (AL, AHLF, and
12
PRHS) and the EMBL International Ph.D. Programme (TB, AHLF, PRHS, AL).
13
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30
A
Cross-striation, solid Z-discs
B
Skeletal myocyte
core regulatory complex
MyoD E12
MASTR
SRF/MEF2
Cross-striation, discontinuous Z-elements
C
Striated effectors
skeletal actin
skeletal troponin C
skeletal troponin I
ZASP/LDB3
Transcription factor
families
bHLH
SRF or
MEF2
(MADS
domain)
Myocardin
(SAP
domain)
Smooth myocyte
Fox
FoxF1
Oblique striation, discontinuous Z-elements
Nkx3.2
Myocardin
SRF/MEF2
GATA6
Smooth ultrastructure
D
Brunet et al. Figure 1
GATA
NK
direct
enhancer
binding
demonstrated
regulation,
direct binding
untested
Cardiac myocyte
FoxC1
Thin myofilament: actin
Thick myofilament: myosin
Dense bodies
Smooth effectors
smooth actin
smooth MLCK
SM22alpha
telokin
caldesmon
Nkx2.5
?
Myocardin
SRF/MEF2
GATA4
?
?
Cardiac effectors
cardiac actin
cardiac MHC
cardiac troponin T
?
demonstrated
co-regulation,
direct binding
untested
1
2
Figure 1. Ultrastructure and core regulatory complexes of myocyte types.
3
(A) Schematic smooth and striated ultrastructures. Electron-dense granules called “dense
4
bodies” separate adjacent myofibrils. Dense bodies are scattered in smooth muscles, but
5
aligned in striated muscles to form Z lines. (B-D) Core regulatory complexes (CRC) of
6
transcription factors for the differentiation of different types of myocytes in vertebrates.
7
Complexes composition from (Creemers et al. 2006; Meadows et al. 2008; Molkentin et
8
al. 1995) for skeletal myocytes, (Hoggatt et al. 2013; Nishida et al. 2002; Phiel et al.
9
2001) for smooth myocytes, and (Durocher et al. 1997; Lee et al. 1998) for
10
cardiomyocytes. Target genes from (Blais et al. 2005) for skeletal myocytes, (Nishida et
11
al. 2002) from smooth myocytes, and (Schlesinger et al. 2011) for cardiomyocytes.
12
31
posted online Jul. 20, 2016;
doi: http://dx.doi.org/10.1101/064881
copyright holder for this preprint (which was not
dpf
11 dpf
1.5 months . TheB
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND
4.0
International license.
AbioRxiv preprint 7first
Somatic
Visceral
muscles
Ventral half
foregut
A
D
muscles
striated
ventral longitudinal
muscles
smooth/striated
foregut musculature
C
midgut
G H
hindgut
ventral oblique
muscles
V
I
3
E
4
J K L
posterior
foregut
muscles
midgut
musculature
P
axochord
D
Dorsal half
M
parapodial
muscles
F
hindgut
musculature
Somatic muscles
Visceral muscles
E
C
G
mitochondrion
foregut
lumen
Z-lines
F
Zlines
Zlines
D
H
I
Z-lines
dense
bodies
foregut
muscles
Zlines
J
K
coelom
dense bodies
longitudinal muscles
circular
muscles
circular muscles
L
gut epithelium
longitudinal muscles
dense bodies
M
dense
bodies
1
Figure 2. Development and ultrastrcture of visceral and somatic musculature in
2
Platynereis larvae and juveniles. (A) Development of visceral musculature. All panels
3
are 3D renderings of rhodamine-phalloidin staining imaged by confocal microscopy.
4
Visceral muscles have been manually colored green and somatic muscle red. Scale bar:
5
50 µm. (B) Schematic of the musculature of a late nectochaete (6 dpf) larva. Body outline
6
modified from (Fischer et al. 2010). Ventral view, anterior is up. (C-M) Electron
7
micrographs of the main muscle groups depicted in B. Each muscle group is shown
8
sectioned parallel to its long axis, so in the plane of its myofilaments. Scale bar: 2 µm.
9
(H) is a close-up of the region in the yellow box in G. (J) shows the dorsal midgut in
10
cross-section, dorsal side up.
11
32
72 hpf
Mef2
Myocardin
KLF5
Actin
GATA456
FoxF
NK3
Calponin*
SM-MRLC
Smooth molecular profile
SM-MHC
MyoD
Titin
Zasp/LBD3
Troponin T
*
Troponin I
B
Striated molecular profile
ST-MRLC
*
SM-MHC
ST-MHC
A
ST-MHC
Annelid longitudinal
striated muscles
Annelid striated
foregut muscles
*
D
*
6 dpf
C
Annelid
oblique/parapodial
striated muscles
Annelid gut
smooth muscles
Vertebrate striated
trunk muscles
Vertebrate striated
esophageal muscles
Vertebrate gut
smooth muscles
Acto- Calcium Striation Transc. Actomyosin response
factors myosin
Expressed
Not expressed
Transcription Acto- Transcription
factors
myosin factors
Expressed in a subset
1
Figure 3. Expression of smooth and striated muscle markers in Platynereis larvae.
2
Animals have been stained by WMISH and observed in bright field Nomarski
3
microscopy. Ventral views, anterior side up. Scale bar: 25 µm. (A-D) Expression patterns
4
of the striated marker ST-MHC and the smooth marker SM-MHC. These expression
5
patterns are representative of the entire striated and smooth effector module (see Figure
6
3-supplements 1 and 3). (E) Table summarizing the expression patterns of smooth and
7
striated markers in Platynereis and vertebrate muscles. (*) indicates that Platynereis and
8
vertebrate Calponin are mutually most resembling by domain structure, but not one-to-
9
one orthologs, as independent duplications in both lineages have given rise to more
10
broadly expressed paralogs with a different domain structure (Figure 7—supplement 3).
11
12
33
A
c. b.
Lifeact-EGFP
H2B-RFP
B
c. b.
Lifeact-EGFP
H2B-RFP
GCaMP6s
D
E
52 μm
61 μm
t0
F
t0+280ms
t0+436 ms
myocytes
1.2
H2B-RFP
50 μm
midgut
t0
0.8
B’
t0+280ms 3 dpf
0.4
H2B-RFP
foregut
51
μm
Contraction speed (s-1)
t0
telotroch
p = 8.5*10-8
0.0
20 μm
A’
FM-464FX
t0
telotroch
6 dpf
G
60
μm
longitudinal
muscle
c. b.
ventral
longitudinal
muscles
C
Somatic striated Visceral smooth
muscles
muscles
t0+1.3s
1
1
31
μm
27
μm
2
2
hindgut
50 μm
6 dpf
1
Figure 4. Contraction speed quantifications of smooth and striated muscles. (A-
2
B) Snapshots of a time lapse live confocal imaging of a late nectochaete larva expressing
3
fluorescent markers. Ventral view of the 2 posterior-most segments, anterior is up.
4
(C) Snapshots of a time lapse live confocal imaging of a 3 dpf larva expressing
5
GCaMP6s. Dorsal view, anterior is up. (D-E) Two consecutive snapshots on the left
6
dorsal longitudinal muscle of the larva shown in C, showing muscle contraction.
7
(F) Quantification of smooth and striated muscle contraction speeds (see Experimental
8
procedures). (G) Snapshot of a time lapse live confocal imaging of a late nectochaete
9
larva. Ventral view, anterior is up. Optical longitudinal section at the midgut level.
10
11
34
A
B
head
pharynx
C Kymograph
line of
interest
longitudinal
fibres
circular
fibres
parapodia
space (x)
Δx
contraction
waves
outline of
the gut
gut
wave propagation
=
speed
7
2
3
4
5
6
n. s.
1
F
Control Brefeldin Atreated
+Brefeldin-A
+2-octanol
Brefeldin Atreated
time (t)
Recurrence of contraction events
n. s.
Number of contractions
per minute
1 2 3 4 5 6
300
200
100
Control
Control
G
FM464-FX
E
p = 5*10-3
0
Wave propagation speed ( m/s)
D
100 μm
Control Brefeldin Atreated
+Brefeldin-A +2-octanol
2 months
400 μm
Δx
Δt
Δt
1
Figure 5. Platynereis gut peristalsis is independent of nervous inputs and dependent
2
on gap junctions. (A) 2 months-old juvenile mounted in 3% low-melting point (LMP)
3
agarose for live imaging. (B) Snapshots of a confocal live time lapse imaging of the
4
animal shown in A. Gut is observed by detecting fluorescence of the vital membrane dye
5
FM-464FX. (C) Kymograph of gut peristalsis along the line of interest in (B).
6
Contraction waves appear as dark stripes. A series of consecutive contraction waves is
7
called a contraction event: here, two contraction waves are visible, which make up one
8
contraction event with a recurrence of 2. (D) Quantification of the propagation speed of
9
peristaltic contraction waves in mock (DMSO)-treated individuals and Brefeldin A-
10
treated individuals (inhibiting neurotransmission). Speed is calculated from kymographs
11
(see Material and Methods). (E,F) Same as in E, but showing respectively the frequency
12
of initiation and the recurrence of contraction events. (G) Representative kymographs of
13
controls, animals treated with Brefeldin A (inhibiting neurotransmission), animals treated
14
with 2-octanol (inhibiting gap junctions), and animals treated with both (N=10 for each
15
condition). 2-octanol entirely abolishes peristaltic waves with or without Brefeldin A.
16
17
35
A outline of the gut B
C
D
serotonin
ac-tubulin
serotonin
combined
neurites
50 μm ac-tubulin
10 μm
ac-tubulin
ac-tub
phalloidin
serotonin
1
Figure 6. The enteric nerve net of Platynereis. (A) Immunostaining for acetylated
2
tubulin, visualizing neurites of the enteric nerve plexus. Z-projection of a confocal stack
3
at the level of the midgut. Anterior side up. (B) Same individual as in A, immunostaining
4
for serotonin (5-HT). Note serotonergic neurites (double arrow), serotonergic neuronal
5
cell bodies (arrow, see D), and serotonergic cell bodies without neurites (arrowhead).
6
(C) Same individual as in A showing both acetylated tubulin and 5-HT immunostainings.
7
Snapshot in the top right corner: same individual, showing both neurites (acetylated
8
tubulin, yellow) and visceral myofibres (rhodamine-phalloidin, red). The acetylated
9
tubulin appears yellow due to fluorescence leaking in the rhodamine channel. (D) 3D
10
rendering of the serotonergic neuron shown by arrow in B.
11
12
36
Slow-contracting group NM/SM-MHC+
Cardiomyocytes
Porifera
Cnidaria
Deuterostomia
striation,
fast
module
Somatic striated
myocytes
Gut myocytes
Protostomia
Protostomia
Vertebrate
Sponge
Nematostella
Annelid
Drosophila Drosophila gut
exopinacocyte slow myocytes cardiomyocyte cardiomyocyte cardiomyocyte
striated
myocytes
SM-MHC
loss
Fast-contracting group ST-MHC+
SM-MHC
loss
SM-MHC
loss
striation,
fast module striation,
fast module
Deuterostomia Deuterostomia
Annelid gut
smooth
myocytes
Vertebrate gut
smooth
myocytes
sm-actin
sm-MELC
Vertebrate
striated
myocytes
Annelid
striated
myocytes
Cnidaria
Planarian
Nematostella
somatic
fast
smooth myocytes myocytes
st-actin
st-MELC
actin
MELC
myocardin
Protostomia
striation
loss
Porifera
Jellyfish
Sponge
striated reticuloapopylocyte
myocytes
striation:
convergent
evolution
MASTR
myocardin
connexin
gap junctions
SM-MRLC
GATA456
?
?
ST-MRLC
MRLC
innexin
gap junctions
SM-MHC
striation:
titin, troponin complex
MyoD
ST-MHC
?
?
change in core regulatory complex
apomere
SM-MHC
module co-option
module loss
species split
cell type split
paralog 1
paralog 2
reciprocal loss of expression
(division of labor)
paralog 2
ancestral
gene
MHC
ST-MHC
paralog 1
gene duplication
1
Figure 7. The evolutionary tree of animal contractile cell types. Bilaterian smooth and
2
striated muscles split before the last common protostome/deuterostome ancestor.
3
Bilaterian myocytes are split into two monophyletic cell type clades: an ancestrally SM-
4
MHC+ slow-contracting clade (green) and an ancestrally ST-MHC+ fast-contracting
5
clade (orange). Hypothetical relationships of the bilaterian myocytes to the SM-MHC+
6
and ST-MHC+ contractile cells of non-bilaterians are indicated by dotted lines (Steinmetz
7
et al. 2012). Apomere: derived set of effector genes common to a monophyletic group of
8
cell types (Arendt et al. 2016). Note that ultrastructure only partially reflects evolutionary
9
relationships, as striation can evolve convergently (as in medusozoans), be co-opted (as
10
in insect gut myocytes or in vertebrate and insect cardiomyocytes), be blurred, or be lost
11
(as in planarians). Conversion of smooth to striated myocytes took place by co-option of
12
striation proteins (Titin, Zasp/LDB3) and of the fast contractile module (ST-MHC, ST-
13
MRLC, Troponin complex) in insect cardiomyocytes and gut myocytes, as well as in
14
vertebrate cardiomyocytes.
15
16
37
foxA
B
foxA
*
C
bra
*
D
hnf4
*
E
*
foregut
A
bra
6 dpf
6 dpf
6 dpf
6 dpf
Brunet et al. Figure 2 - supplement 1
hindgut
midgut
foxA
bra+foxA
hnf4
1
Figure 2—figure supplement 1. Gut patterning in Platynereis 6 dpf larvae. (A-D)
2
WMISH for gut markers. (A) Ventral view, anterior is up. (B-D) Left lateral views,
3
anterior is up, ventral is right. (E) Schematic of gut patterning in Platynereis late
4
nectochaete larvae. Scale bar: 50 µm.
5
6
38
B
zasp
C
titin
D
troponin I
E
troponin T
48 hpf
A
ST-MRLC
G
H
I
J
K
L
M
N
O
6 dpf
72 hpf
EF
P
Q
Q
R
l
U
ST-MHC
S
ST-MRLC
T
troponin I
V
troponin T
W
titin
zasp
1
Figure 3—supplement 1. Expression of striated muscle markers in Platynereis
2
larvae. (A-O) Larvae stained by WMISH and observed in bright field Nomarski
3
microscopy. Ventral views, anterior side up. Scale bar is 20 µm for 48 hpf and 25 µm for
4
the two other stages. (P) Foregut musculature visualized by rhodamine-phalloidin
5
fluorescent staining. Z-projection of confocal planes. Ventral view, anterior side up.
6
Scale bar: 20 µm. (Q) TEM micrograph of a cross-section of the foregut. Foregut muscles
7
are colored green, axochord orange, ventral oblique muscles pink, ventral nerve cord
8
yellow. Inset: zoom on the area in the red dashed box with enhanced contrast to visualize
9
oblique striation. Scale bar: 10 µm. (R-W) WMISH for striated muscle markers
10
expression in the foregut observed in Nomarski bright field microscopy, ventral views,
11
anterior side up. Scale bar: 20 µm.
12
13
39
Axochord
titin
zasp
ST-MHC
ST-MRLC
DAPI
**
**
* *
*
* *
*
**
*
** *
*
*
DAPI
*
*
*
*
*
*
*
*
** *
*
*
Oblique muscles
DAPI NBT/BCIP
*
*
*
*
*
*
*
*
*
troponin I
*
*
*
*
*
*
myoD
troponin T
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*
*
* *
*
*
*
*
*
*
*
*
*
*
*
*
**
*
** *
*
*
**
*
*
* *
*
* *
** *
*
* *
*
* *
** *
*
** *
*
* **
*
**
* **
*
**
*
*
*
*
*
*
** * *
*
* * *
*
*
**
*
* **
*
*
*
*
*
*
* *
* **
*
*
*
**
*
*
* *
**
*
*
*
* *
*
*
*
*
*
*
* *
*
*
*
*
*
* *
* *
* **
*
*
*
*
*
**
*
*
*
*
*
*
* * *
*
*
*
**
*
*
*
** * *
*
*
*
*
*
*
*
**
* **
**
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
* *
*
*
*
*
*
*
*
DAPI NBT/BCIP
*
*
*
**
*
*
*
DAPI
*
*
*
Parapodial muscles
*
*
*
*
*
*
*
DAPI NBT/BCIP
*
*
*
*
DAPI
*
*
* *
* *
*
*
*
*
*
*
*
Antennal muscles
DAPI NBT/BCIP
*
**
*
*
*
DAPI
*
*
*
Longitudinal muscles
*
*
*
*
*
*
*
*
*
* *
* *
*
*
DAPI NBT/BCIP
*
** *
*
*
*
**
*
*
*
**
*
*
** *
*
*
*
**
*
*
*
1
Figure 3—supplement 2. Expression of striated muscle markers in the 6 dpf
2
Platynereis larva. Animals have been stained by WMISH and observed by confocal
3
microscopy (DAPI fluorescence and NBT/BCIP 633 nm reflection). All striated effector
4
genes are expressed in all somatic muscles examined. The transcription factor myoD is
5
detectable in the axochord and in ventral longitudinal muscles, but not in other muscles.
6
7
40
SM-MRLC
72 hpf
A
B
GATA456
C
l. d.
E
F
*
D
foxF
*
*
*
*
6 dpf
calponin
*
l. d.
G
*
72 hpf
H
*
forming
NK3 gut muscles
*
M
N
dlm
dlm
dlm
DAPI FM-464FX
phalloidin
heart
O
SM-MHC coloc.
t.f.
l.f.
72 hpf
K foxF
GATA456 calponin
72 hpf
heart
coelom
gut
muscles
SM-MHC
DAPI
heart
intrinsic
muscles
gut
muscles
gut
gut
P
heart
dlm
gut
NK3
actin coloc.
gut
muscles
gut
heart
tube
actin
Q
dorsal longitudinal
muscles
coelom
intrisic
muscles
heart tube
2 months - heart tube
L
SM-MHC
DAPI
t.f.
l.f.
72 hpf
DAPI FM-464FX
phalloidin
J
I
ST-MHC
DAPI
intrisic
muscles
striated musculature ST-MHC+
gut
smooth musculature SM-MHC+
blood
epidermis
coelomic lining
1
Figure 3—supplement 3. Expression of smooth muscle markers in Platynereis
2
larvae. (A-H) Animals are stained by WMISH and observed by Nomarski bright field
3
microscopy. Ventral views, anterior side up. Yellow arrows: expression in the foregut
4
mesoderm. White dashed lines: outline of the midgut and hindgut (or their anlage at 3
5
dpf). Asterisk: stomodeum. (I) Schematic drawing of a 3 dpf larva (ventral view, anterior
6
is up) showing the forming midgut and hindgut (dotted line) and the forming visceral
7
myofibres in green (compared to double WMISH panels J and K). (J,K) Animals are
8
stained by double WMISH and observed by confocal microscopy (cyanine 3 precipitate
9
fluorescence for actin, NBT/BCIP precipitate reflection for other genes). Ventral views,
10
anterior side up. Abbreviations: t.f.: transverse fibres; l.f.: longitudinal fibres. (L-Q)
11
Molecular profile of the pulsatile dorsal vessel. All panels show 2 months-old juvenile
12
worms. (L,M) Maximal Z-projections of confocal stacks. Dorsal view, anterior is up. (L)
13
Dorsal musculature of a juvenile Platynereis dumerilii individual visualized by
14
phalloidin-rhodamine (green) together with nuclear (DAPI, blue) and membrane (FM-
15
464FX, red) stainings. The heart tube lies on the dorsal side, bordered by the somatic
16
dorsal longitudinal muscles (dlm). (M) Expression of SM-MHC in the heart tube
17
visualized by WMISH. (N) Virtual cross-section of the individual shown in A. Dorsal
18
side up. Note the continuity of the muscular heart tube with gut musculature. (O) Virtual
19
cross-section of the individual shown in B, showing continuous expression of SM-MHC
20
in the heart and the midgut smooth musculature. Note the similarity to the NK4/tinman
21
expression pattern documented in (Saudemont et al. 2008). (P) Virtual cross-section on
22
an individual stained by WMISH for ST-MHC expression. Note the lack of expression in
23
the heart, while expression is detected in intrinsic muscles that cross the internal cavity to
41
1
suspend internal organs, and in dorsal longitudinal muscles (dlm). (Q) Schematic cross-
2
section of a juvenile worm (dorsal side up) showing the shape, connections and molecular
3
profile of the main muscle groups. Scale bar: 30 µm in all panels.
4
5
42
B
SM-MRLC
Smooth muscle markers
A
C
calponin
D GATA456
E
foxF
G
ST-MHC
H
J
troponin I
K
SM-MHC
foregut
*
midgut
6 dpf
Striated muscle markers
F
I
ST-MRLC
titin
zasp
troponin T
1
Figure 3—supplement 4. Molecular profile of midgut muscles in the 6 dpf larva. All
2
panels are Z-projections of confocal planes, ventral views, anterior side up. Blue: DAPI,
3
red: NBT/BCIP precipitate. White dashed line: midgut/hindgut, yellow dashed ellipse:
4
stomodeum. (A-E) Smooth markers expression. White arrows indicate somatic
5
expression of GATA456 in the ventral oblique muscles. (F-K) Striated markers
6
expression; none of them is detected in any gut cell. White arrows: somatic expression.
7
Scale bar: 20 µm in all panels.
8
9
43
*
actin
48 hpf
*
A
dlm
24 hpf B
dlm
24 hpf C
D dlmvlm
vlm
48 hpf
E
am
a
72 hpf
F
dlm
a vom
lom
vom
lom
vc
*
vc
G
pm
lom
myocardin
*
actin
mef2
6 dpf H
6 dpf J
I
f.m.
f.m.
6 dpf L
K
f.m.
f.m.
f.m.
midgut
midgut
M Oblique muscles
DAPI
midgut
DAPI NBT/BCIP
DAPI
DAPI NBT/BCIP
Oblique muscles
DAPI
DAPI NBT/BCIP
*
*
*
*
*
*
*
*
*
*
*
*
vlm
*
*
* **
*
DAPI
DAPI NBT/BCIP
r.a.
Oblique muscles
DAPI
DAPI NBT/BCIP
ach
*
*
vlm
*
* **
midgut
midgut
midgut
ach
*
72 hpf
*
*
**
*
*
**
*
* *
*
* *
*
DAPI
*
*
*
*
*
*
DAPI NBT/BCIP
*
*
*
*
*
*
1
Figure 3—supplement 5. General muscle markers are expressed in both smooth and
2
striated muscles. All panels show gene expression visualized by WMISH. (A-F) actin
3
expression. (A-B) bright field micrographs in Nomarski optics. (A) is an apical view, (B)
4
is a ventral view. Abbreviations: dlm, dorsal longitudinal muscles; vc, ventral
5
mesodermal cells, likely representing future ventral musculature. (C-F) 3D rendering of
6
confocal imaging of NBT/BCIP precipitate. (C,E) ventral views, anterior side up. (D,F)
7
ventrolateral views, anterior side up. Abbreviations are as in Figure 1. (G-M) Z-
8
projections of confocal stacks. Blue is DAPI, red is reflection signal of NBT/BCIP
9
precipitate. (G-L) Ventral views, anterior side up. White dashed line: midgut, yellow
10
ellipse: foregut. White arrows: somatic muscle expression. Abbreviations: f.m.: foregut
11
muscles; r.a.: reflection artifact. (M) Expression in individual somatic muscles. Scale bar:
12
20 µm in all panels.
13
14
44
Evolution of somatic striated CRC
Vertebrate trunk
striated myocytes
Platynereis trunk striated
longitudinal myocytes
MyoD E12
MyoD E12
MASTR
Nau
Myocardin
SRF/MEF2
Drosophila trunk
striated myocytes
Da
hlh-1 hnd-1
Striated
effectors
MEF2
MEF2
Caenorhabditis trunk
striated myocytes
Striated
effectors
UNC-120
Striated
effectors
Striated
effectors
MyoD E12
Myocardin
SRF/MEF2
Ancestral somatic
striated CRC
Striated
effectors
Gut myocytes branch
Vertebrate gut
smooth myocyte
Platynereis gut
smooth myocyte
FoxF1
Nkx3.2
GATA6
Drosophila gut
striated myocyte
FoxF
Myocardin
NK3
SRF/MEF2
GATA456
MEF2
Smooth contractile and
differentiation effectors
Platynereis
cardiomyocyte
GATA4
MEF2
Gut myocyte
differentiation
effectors
NK4
Myocardin
Nkx2.5
SRF/MEF2
Striated
effectors
Striated contractile
effectors
Myocardin
FoxC
Ancestral gut
smooth CRC
NK4
Myocardin
SRF/MEF2
GATA4/5/6
Smooth contractile effectors
Differentiation effectors
(morphogenesis, innervation, adhesion)
Ancestral
cardiac CRC
Smooth
effectors
Fox
NK
Myocardin
SRF/MEF2
GATA4/5/6
Ancestral visceral
smooth CRC
Smooth contractile effectors
Differentiation effectors
MEF2
Striated
effectors
striation
striation
SRF/MEF2
Pannier
Smooth
effectors
FoxF
NK3
tinman
Myocardin
?GATA4/5/6 SRF/MEF2
striation
GATA4/5/6
Drosophila
cardiomyocyte
FoxC1
bagpipe
SRF/MEF2
Cardiomyocytes branch
Vertebrate
cardiomyocyte
biniou
Myocardin
Smooth contractile and
differentiation effectors
Evolution of visceral smooth CRC
1
Figure 7—supplement 1. Evolution of myogenic Core Regulatory Complexes (CRC)
2
in Bilateria. Transcription factor families are depicted as in Figure 1. Direct contact
3
indicates proven binding. Co-option of the fast/striated module happened on three
4
occasions: in Drosophila gut myocytes, and in cardiomyocytes of both vertebrates and
5
Drosophila. Note that in both cases, composition of the CRC was maintained in spite of
6
change in the effector module. In insect gut myocytes, replacement of the smooth by the
7
striated module entailed a split of the CRC, with the ancient smooth CRC still controlling
8
conserved differentiation genes (involved in adhesion, morphogenesis, axonal guidance,
9
or formation of innexin gap junctions), while the striated contractile cassette is
10
downstream Mef2 alone (Jakobsen et al. 2007b). It is less clear whether a similar split of
11
CRC took place in striated cardiomyocytes.
12
13
45
A
Deuterostomia
Protostomia
peer-reviewed)
is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International
license.
Tardigrada
Nematoda
Priapulida
Annelida
Arthropoda
Ecdysozoa
Mollusca
Chaetognatha
Chordata
Echinodermata
Spiralia
1.00
1.00
0.74
0.73
0.97
0.97
1.00
Ultrastructure of midgut muscles
Smooth
One-sarcomere
Striated
0.82
0.83
0.54
0.82
0.89
Insecta
0.90
0.77
0.77
0.69
Peripatopsis
Arenicola
Siboglinum
0.80
Panarthropoda
0.66
0.61
0.53
Helobdella
Eisenia
0.62
0.88
Ecdysozoa
Annelida
Mytilus
Helix
Rossia
Lepidopleurus
Meiomenia
Mollusca
Hemithris
Cephalodiscus
Rhabdopleura
Hemichordata
Saccoglossus
Cephalochordata
Urochordata
Vertebrata
Chordata
Spiralia
Crustacea
Deuterostomia
Flabelliderma
B
Chelicerata
0.98
0.62
0.79
0.73
0.63
0.56
Striated heart
myocytes
Smooth heart
myocytes
1
Figure 7—supplement 2. Ancestral state reconstructions of the ultrastructure of
2
midgut/hindgut
3
reconstruction, of midgut smooth muscles in Bilateria. Ancestral states were inferred
4
using Parsimony and Maximum Likelihood (ML) (posterior probabilities indicated on
5
nodes). Character states from: Chordata (Marieb & Hoehn 2015), Echinodermata (Feral
6
& Massin 1982), Chaetognatha (Duvert & Salat 1995), Mollusca (Royuela et al. 2000),
7
Annelida (Anderson & Ellis 1967), Priapulida (Carnevali & Ferraguti 1979), Nematoda
8
(White 1988), Arthropoda (Goldstein & Burdette 1971), and Tardigrada (Shaw 1974).
9
(B) Distribution, and ancestral state reconstruction, of cardiomyocyte ultrastructure in
10
Bilateria. Ancestral states were inferred using Parsimony and ML (posterior probabilities
11
indicated on nodes). Note that, due to the widespread presence of striated cardiomyocytes
12
in bilaterians, the support value for an ancestral smooth ultrastructure in the ML method
13
remain modest (0.56). This hypothesis receives independent support from the comparison
14
of CRCs (Figure 1B, Figure 7—supplement 1) and developmental data (see Discussion).
15
Character states follow the review by Martynova (Martynova 2004; Martynova 1995) and
16
additional references for Siboglinum (Jensen & Myklebust 1975), chordates (Hirakow
17
1985), Peripatopsis (Nylund et al. 1988), arthropods (Tjønneland et al. 1987), Meiomenia
18
(Reynolds et al. 1993), and Lepidopleurus (Økland 1980). In Peripatopsis and
19
Lepidopleurus, some degree of alignment of dense bodies was detected (without being
20
considered regular enough to constitute striation), suggesting these might represent
21
intermediate configurations.
and
heart
myocytes.
(A) Distribution,
and
ancestral
state
22
46
A
Homo sapiens
Platynereis dumerilii
Hsa-calponin1
Pdu-calponin
Hsa-calponin2
Pdu-transgelin1
Hsa-calponin3
Pdu-transgelin2
C
am
auhm
Drosophila melanogaster
Hsa-transgelin1
mp20
Hsa-transgelin2
Hsa-transgelin3
Caenorhabditis elegans
vlm
ach
chd64
vom
Domains
Calponin homology
unc-87
Calponin repeats
B
Branchiostoma-floridae-calponin
Danio-transgelin
0,9907
Danio-transgelin2
Homo-transgelin2
1
Rattus-transgelin2
1
Rattus-transgelin
1
Homo-transgelin
Homo-transgelin3
Mus-calponin2
1
Homo-calponin2
1
Xla-calponin2B
Latimeria-calponin3-isoformX1
1
Latimeria-calponin3-isoformX2
0,5786
Homo-calponin3
1
Oryzias-calponin3-like
Danio-calponin3b
0,99730,9787
0,9973Danio-calponin3a
Astyanax-calponin3-like
Danio-calponin2
0,5846
Xiphophorus-calponin1-like
1
Takifugu-calponin1-like
Meleagris-calponin1
1
0,5959
Gallus-calponin1
0,52
Chrysemys-calponin1
0,5959
Mus-calponin1
1 Sus-calponin2
0,9794
Sus-calponin3
Homo-calponin1
Ceratitis-capitata-myophilin-likeX1
0,998
Drosophila-chd64
1
Musca-domestica-myophilinX1
1
Riptortus-pedestris-calponin-transgelin
0,9734
Daphnia-pulex-DAPPUDRAFT_308120
0,8702
Nasonia-vitripennis-myophilin
0,98
Bombyx-mori-transgelin
0,9927
Pdu-transgelin1
1
Pdu-transgelin2
0,5473
Echinococcus-myophilin
0,9953
1
Drosophila-mp20
Metaseiulus-occidentalis-mp20-like
Solen-grandis-calponin-like-protein
0,5839
Mytilus-galloprovincialis-calponin-like
0,7603
Helobdella-robusta-HELRODRAFT_186180
1
Capitella-teleta-CAPTEDRAFT_17776
1
1
Cerebratulus-lacteus-calponin
Capitella-myophilin
0,715
Pdu-calponin
Lottia-gigantea-LOTGIDRAFT_149649
Aplysia-californica-myophilin-like
Crassostrea-gigas-Mp20
Transgelin
clade
0,8256
1
Deuterostomia
Calponin
clade
0,7643
1
1
0,7716
Protostomia
transgelin1
DAPI
D
midgut
transgelin1
DAPI
0.2
Brunet et al. - Figure 7 supplement 3
1
Figure 7—supplement 3. Domain structure, phylogeny and expression patterns of
2
members of the calponin gene family. (A) Domain structure of calponin-related
3
proteins in bilaterians. Calponin is characterized by a Calponin Homology (CH) domain
4
with several calponin repeats, while Transgelin is characterized by a CH domain and a
5
single calponin repeat. In vertebrates, Calponin proteins are specific smooth muscle
6
markers, and the presence of multiple CH repeats allows them to stabilize actomyosin
7
(Gimona et al. 2003). Transgelins, with a single calponin repeat, destabilize actomyosin
8
(Gimona et al. 2003), and are expressed in other cell types such as podocytes (Gimona et
9
al. 2003), lymphocytes (Francés et al. 2006), and striated muscles (transiently in mice (Li
10
et al. 1996) and permanently in fruit flies (Ayme-Southgate et al. 1989)). (B) Maximum
11
Likelihood phylogeny of the calponin/transgelin family based on alignment of the CH
12
domain. Paralogs with calponin and transgelin structures evolved independently in
13
vertebrates and Platynereis (Pdu, in red squares). (C) Expression patterns of Pdu-
14
transgelin1. Scale bar: 25 µm. As in vertebrates and insects, transgelin1 is not smooth
15
myocyte-specific, but also detected in striated myocytes.
16
17
47
A
CapsaCalmo
bioRxiv
preprint first posted online Jul. 20, 2016; doi: http://dx.doi.org/10.1101/064881. The copyright holder for this preprint (which was not
BosCalmo
0.419
DmeCalmo
ThtrCalmo
0.763
Fungi
Fungi MRLC
Fungi MRLC
FalbaMRLC
0.747 Sponge MRLC
0.99
Sponge MRLC
OscMRLC
1
0.87
0.97 Cnidarian MRLC
0.997
0.291
0
Porifera
Cnidaria
BflMRLCb
Striated MRLC
0.718
CteMRLCa
0.99
Platynereis-ST-MRLC
0.77
0.628
0.99
RypaRLC
Mollusc striated MRLC
0.923
0.48
0.74
Arthropod muscle MRLC
1
0.99
0.88
1
Arthropod/nematode muscle MRLC
0.006
Vertebrate striated MRLC
0.86
1
0.8
SpuMRLCa
0.678
SkoMRLCb
SpuMRLCb
0.715
SpuMRLCc
0.83
1 Nematode non-muscle MRLC
Smooth MRLC
0.77
SkoMRLCa
0.86
0.96 Insect non-muscle MRLC
Amphioxus MRLCa
0.71
0.87
Vertebrate-smooth/non-muscle MRLC
0.98
Platynereis-SM-MRLC
0.833
HelobdMRLC
Mollusc smooth MRLC
0.9
BatdenMRLC
Fungi
0.606
MonveMRLC
0.4
Dme-biniou
B
Pdu-FoxF
Cgi-LBD3
C
1
Aca-LBD3
0.9956
Cte-FoxF
0.9878
1
1
Patvu-FoxF
1
Hro-LBD3
1
Brafl-FoxF
Cte-LBD3
1
Mus-FoxF2
1
1
Hsa-FoxF2
1
1
Pdu-ZASP/LBD3
Mus-FoxF1
GgaLBD3X12
Xenla-FoxF1B
1
1
1
Xenla-FoxF1A
HSALBD3X14
0.9612
1
Hsa-FoxF1
DreCyphX3a
Mus-FoxQ1
1
Cte-FoxQ1
0.2
Brunet et al. Supplementary Figure 1
Nve-LBD3
0.2
Bilateria
0.704
D
bioRxiv preprint first posted online Jul. 20,
2016; doi: http://dx.doi.org/10.1101/064881
. The copyright holder for this preprint
(which was not
Cte-Titin
Cel-MEF2
HSA-MYH7
0
DANRE-AMHC
0
DANRE-MYH1
1
F
Tt-Mef2
0.99
1
HSA-MYH1
Dme-MEF2
1
1
DME-ST-MHC
0.992
G
1
Dre-TitinA
1
1
Hsa-MEF2D
Dre-TitinB
1
1
Xenla-MEF2D
BGANMMHCX3
SkoTitin
NemvMef2X1
Bfl-TropT
Cint-TropT-X5
0.986
Hsa-Titin
1
DanreMEF2D
SKOMYH10X2
0.2
1
Danre-MEF2C
0.94
ACANMMHCX1
1
Mus-Titin
0.5876
MUS-MYH11
Platynereis-SM-MHC
0.984
Cel-Titin
Xenla-MEF2C
1
LPO-NMMHC
0.989
PolisTitin
0.79
0.94
HSA-MYH11
DME-NM-MHC
0.999
1
Hsa-MEF2C
GGA-MYH11
1
Limu-Titin
DanreMEF2A
1
DANREMYH11
0.999
PrcauTitin
XenlaMEF2A
MUS-MYH9
1
1
0.91
HSA-MYH9
Aca-titin
Cgig-Titin
1
Octo-Titin
Hsa-MEF2A
1
MUS-MYH10
0
1
DanreMEF2B
1
0
0.6208
0.6086
1
0.85
HSA-MYH10
1
Lgi-Titin
1
Hsa-MEF2B
DME-M-MHC
1
Hro-Titin
Pdu-titin
Platynereis-ST-MHC
0.994
1
1
Pdu-mef2
HSA-MYH8
1
1
E
SpurTwitch
0.2
H
Onchocerca-calponin-like
1
0.2
Calponin/transgelin clade
0.9996
Hsa-TropI2
0.9997
Oryctolagus-TropI2
Dre-TropT-cardiac
0.9
0.9982
0.34
0.8288
Hsa-TropT-skeletal
0.738
0.962
Xla-TropI3
Mus-TropT-cardiac
0.6167
0.886
Rattus-TropI3
0.7829
Drome-TropT
0.9999
Hsa-TropI3
Gga-TropI3
0.9998
Cte-TropT
0.849
Mus-TropI3
0.9999
Hsa-TropT-cardiac
0.986
Gga-TropI2
Hsa-TropI1
Coturnix-TropI3
Helro-TropT
1
Dm-TropI
1
Astacus-TropI
0.227
Pdu-TropT
0.872
Ce-TropI3
1
1
Cgi-TropT-X8
0.9998
0.79
0.845
0.9991
Linan-TropT
Bbe-MyoD2
0.3803
I
Bbe-MyoD1
Mus-MYKL2
0.8647
Hsa-MKL1
0.6341
1
Hsa-MYOD1
Mus-MYKL1
Xenla-MRTFA
1
0.2914
Hsa-Myf5
Gga-MYCD
Xenla-Myf5
Hsa-MYCD
1
0.5938
Dre-Myf5
0.4319
1
Mus-MYCD
0.6741
Mus-Myog
Xenla-MYCD
0.2719
HSA-Myf6
0.6148
Pdu-TropI
1
0.2224
Xenla-MyoD
0.3348
Chlamys-TropI
0.4
Hsa-MYLK2
J
DanreMyoD
0.5046
Ce-TropI1
Ce-TropI4
Sma-TropT
0.4
Ce-TropI2
0.9678
1
Pdu-MYCD
Spur-MyoD1
0.9302
0.5143
Sacko-MyoD
Lottia-gigantea-LOTGIDRAFT_111577
0.7073
Crassostrea-gigas-MKL-myocardin-like-protein-2
Tt-MyoD
1
0.5314
Daphnia-pulex-DAPPUDRAFT_309984
Pdu-MyoD
0.9989
0.5938
Spur-MyoD2
0.5326
0.8282
Drosophila-melanogaster-Myocardin-related-transcription-factor-isoform-H
Stegodyphus-mimosarum-MKL-myocardin-like-protein-2
Dme-nau
0.07
0.2
CapsaCalmo
BosCalmo
DmeCalmo
ThtrCalmo
0,291
MucorMRLC
LichcoMRLC
0,988
0,482
0,006
0,878
0,987
0.4
0,715
SpuMRLCa
0,678
SkoMRLCb
SpuMRLCb
SpuMRLCc
AscMRLC
WuchMRLC
1
CelMLC4
AncyMRLC
0,77
SkoMRLCa
HarpeMRLCn
0,83
0,87
AnopMRLCnm
BombyxMRLC
DanaMRLCsm
DmeMRLCnm
CeraMRLCnm
BflMRLC
1 BflMRLCa
Callorhinc
CalMRLC12B
LepiMRLC
1
GgaMRLCsm
0,868
RatMRLCsm
HsaMRLCsm
PterMRLCsm
BosMRLCsm
XentrMRLCs
TtdnMRLC
FuguMRLC9
AstyMRLC9
IctaMRLCsm
OryzMRLCsm
OreoMRLCsm
0,833
Pdu-SM-MRLC
HelobdMRLC
CrassosMRL
0,343
LgiMRLCb
0,901
ApcalMRLC
0,965
0,791
0,718
0,768
LeucMRLC6
SyconMRLCa
0,747
LeucMRLC19
LeucMRLC21
1
SyconMRLCb
LeucMRLC23
EphmuMRLC
0,988AmquMRLC
OscMRLC
0,87
MontaMRLC3
0,971
1
NveMRLCb
MetseMRLCb
0,971
0,997
FalbaMRLC
0,738
BatdenMRLC
MonveMRLC
AllomaMRLC
0,763 SalpunMRLC
0,704
1
0,606
K
0,864
0,923
0,628
0,991
Pdu-ST-MRLC
1 PlmaMRLC
AirMRLCm
ScolopMRLC
TriboMRLC
0,797
0,997
ArtfraMRLC
0,422
DaphMRLC
1
1
DaphMRLCb
BmorMRLCb
0,596
DmeMRLCm
0,98
AnopMRLCb
TrichiMRLC
1
LoaMRLC1
Necator-am
0,922
CelMLC1
CelMLC2
MusMRLCat
GgaMRLCsk
0,989
HsaMRLCsk
RbMRLCsk
0,904
RatMRLCsk
GgaMRLChe
0,595
HsaMRLCve
RatMRLCve
0,951
BflMRLCb
LgiMRLCd
LgiMRLCa
PfuMRLC
RypaRLC
CteMRLCa
1
Supplementary Figure 1. Phylogenetic trees of the markers investigated.
2
(A) Simplified Maximum Likelihood (ML) tree for Myosin Regulatory Light Chain (full
3
tree in panel M), rooted with Calmodulin, which shares an EF-hand calcium-binding
4
domain with MRLC. (B) ML tree for FoxF, rooted with FoxQ1, the probable closest
5
relative of the FoxF family (Shimeld et al. 2010). (C) MrBayes tree for bilaterian
6
ZASP/LBD3, rooted with the cnidarian ortholog (Steinmetz et al. 2012). (D) ML tree for
7
bilaterian Myosin Heavy Chain, rooted at the (pre-bilaterian) duplication between smooth
8
and striated MHC (Steinmetz et al. 2012). (E) MrBayes tree for Mef2, rooted by the first
9
splice isoform of the cnidarian ortholog (Genikhovich & Technau 2011). (F) MrBayes
10
tree for Titin, rooted at the protostome/deuterostome bifurcation (Titin is a bilaterian
11
novelty). (G) MrBayes tree for Troponin T, rooted at the protostome/deuterostome
12
bifurcation (Troponin T is a bilaterian novelty). (H) MrBayes tree for Troponin I, rooted
13
by the Calponin/Transgelin family, which shares an EF-hand calcium-binding domain
14
with Troponin I. (I) MrBayes tree for MyoD, rooted at the protostome/deuterostome
15
bifurcation (MyoD is a bilaterian novelty). (J) MrBayes tree for Myocardin, rooted at the
16
protostome/deuterostome bifurcation (the Drosophila myocardin ortholog is established
17
(Han et al. 2004)). (K) Complete MRLC tree.
18
Species names abbreviations: Pdu: Platynereis dumerilii; Xenla: Xenopus laevis; Mus:
19
Mus musculus; Hsa: Homo sapiens; Dre: Danio rerio; Gga: Gallus gallus; Dme:
20
Drosophila melanogaster; Cte: Capitella teleta; Patvu: Patella vulgata; Brafl:
21
Branchiostoma floridae; Nve or Nemv: Nematostella vectensis; Acdi: Acropora
22
digitifera; Expal: Exaiptasia pallida; Rat: Rattus norvegicus; Sko: Saccoglossus
23
kowalevskii; Limu or Lpo: Limulus polyphemus; Trib or Trca: Tribolium castaneum;
48
1
Daph: Daphnia pulex; Prcau: Priapulus caudatus; Cgi or Cgig: Crassostrea gigas; Ling
2
or Linan: Lingula anatina; Hdiv: Haliotis diversicolor; Apcal or Aca: Aplysia
3
californica; Spu: Strongylocentrotus purpuratus; Poli or Polis: Polistes dominula; Cin or
4
Cint: Ciona intestinalis; Hro: Helobdella robusta; Bos: Bos taurus; Capsa: Capsaspora
5
owczarzaki; Thtr: Thecamonas trahens; Lpo: ; Bga: Biomphalaria glabrata; Cel:
6
Caenorhabditis elegans; Tt: Terebratalia transversa; Octo: Octopus vulgaris; Sma:
7
Schmidtea mediterranea; Bbe: Branchiostoma belcheri, Batden: Batrachochytrium
8
dendrobaditis; Monve: Mortierella verticillata; Alloma: Allomyces macrogynus; Salpun:
9
Spizellomyces punctatus; Mucor: Mucor racemosus; Lichco: Lichtheimia corymbifera;
10
Ephmu: Ephydatia muelleri; Sycon: Sycon ciliatum; Amqu: Amphimedon queenslandica;
11
Osc: Oscarella lobularis; Metse: Metridium senile; Pfu: Pinctada fucata; Rypa: Riftia
12
pachyptila; Plma: Placopecten magellanicus; Air: Argopecten irradians; Scolop:
13
Scolopendra gigantea; Artfra: Artemia franciscana; Bmor: Bombyx mori; Loa: Loa loa;
14
Necator-am: Necator americanus; Trichi: Trichinella spiralis; Asc: Ascaris lumbricoides;
15
Wuch: Wucheria bancrofti; Ancy: Ancylostoma duodenale; Callorinc: Callorhinchus
16
milii; Dana: Danaus plexippus; Anop: Anopheles gambiae; Asty: Astyanax mexicanus;
17
Oreo: Oreochromis niloticus; Icta: Ictalurus punctatus.
18
19
49

1 The evolutionary origin of bilaterian smooth and striated

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