Modular co-option of cardiopharyngeal genes during non-embryonic myogenesis

Background In chordates, cardiac and body muscles arise from different embryonic origins. In addition, myogenesis can be triggered in adult organisms, during asexual development or regeneration. In non-vertebrate chordates like ascidians, muscles originate from embryonic precursors regulated by a conserved set of genes that orchestrate cell behavior and dynamics during development. In colonial ascidians, besides embryogenesis and metamorphosis, an adult can propagate asexually via blastogenesis, skipping embryo and larval stages, and form anew the adult body, including the complete body musculature. Results To investigate the cellular origin and mechanisms that trigger non-embryonic myogenesis, we followed the expression of ascidian myogenic genes during Botryllus schlosseri blastogenesis and reconstructed the dynamics of muscle precursors. Based on the expression dynamics of Tbx1/10, Ebf, Mrf, Myh3 for body wall and of FoxF, Tbx1/10, Nk4, Myh2 for heart development, we show that the embryonic factors regulating myogenesis are only partially co-opted in blastogenesis, and that markers for muscle precursors are expressed in two separate domains: the dorsal tube and the ventral mesenchyma. Conclusions Regardless of the developmental pathway, non-embryonic myogenesis shares a similar molecular and anatomical setup as embryonic myogenesis, but implements a co-option and loss of molecular modules. We then propose that the cellular precursors contributing to heart and body muscles may have different origins and may be coordinated by different developmental pathways. Electronic supplementary material The online version of this article (10.1186/s13227-019-0116-7) contains supplementary material, which is available to authorized users.


BACKGROUND
Musculature is a tissue specialized in contraction shared among all eumetazoans. Its cellular components contain molecular structures based on actomyosin and an array of accessory proteins which allows contractility 1 . Myogenesis operates through a progressive activation of transcription factors organized in a hierarchical and modular network that drive cell fate specification, and is followed by specific cell behaviors that lead to muscle differentiation and organization 2 . For example, trunk skeletal muscles of vertebrates develop from somites and are determined by the expression of the paired box transcription factor Pax3 3 , whereas cardiac, pharyngeal 4 , and possibly also esophagus striated muscles 5 originate from cardio-pharyngeal mesoderm and are determined by T-Box1. A common origin of heart and pharyngeal muscles from the cardio-pharyngeal field predates vertebrate evolution 4,6-8 and has been hypothesized to be a chordate synapomorphy 9,10 .
In metazoans, muscles can develop during embryogenesis and during non-embryonic development of adult organisms, i.e. regeneration and asexual development. The myogenic process may be triggered by populations of multi-or unipotent stem cells [11][12][13][14] . Post-embryonic myogenesis also occurs in species with indirect development and complex life cycles 15 . These animals undergo drastic changes between their larval and adult bauplan, such as during metamorphosis, and the musculature can radically change architecture within the same organism between different life stages 16,17 .
With their bi-phasic life history and asexually reproducing colonial species, ascidians (Tunicata) offer great opportunities to study the development of different muscle architectures in different life stages 18 . From the fertilized egg, a stereotyped development and largely determinative embryogenesis 19 leads to the formation of a planktonic larval body. In the larval tail, bands of mono-nucleated myocytes are arranged in a striated fashion 20 and express a myosin heavy chain, specific to embryonic muscles 21,22 . When the larva settles on a substrate and metamorphoses into a sessile filter-feeding adult, the sarcomeric arranged musculature gets resorbed along with the tail 23 and non-striated circular and longitudinal body muscles form along the mantle of the organism, together with the cardiac muscles 20,24 . The adult body wall muscle are described as smooth (non-striated) muscles, which apparently evolved due to a loss of sarcomeric organization, probably in order to cope with a sessile lifestyle, which requires slow contractions for the fine tuning of water inflow 20 . The body and heart muscles express two specific, post-metamorphic myosins: myosin heavy chain 3 and myosin heavy chain 2 22 , respectively.
During embryonic development of solitary ascidians, maternal deposition of the zinc finger family member Zic-r.a (Macho-1) is essential for early muscle specification 25 . Zic-r.a activates the expression of the T-box transcription factor Tbx6, necessary to induce the tail muscles 26 . Zic-r.a and Tbx6 together with beta-catenin define the cardio-pharyngeal field by activating the bHLH regulatory gene mesodermal posterior (Mesp) 27 . The cardio-pharyngeal field, a.k.a. the trunk ventral cells (TVCs) in Ciona, give rise to both the heart 28 and part of the adult ascidian body musculature. The expression of the homeobox transcription factor Nk4, orthologue to tinman/Nkx2-5, in TVCs antagonizes Tbx1/10 and promotes the cardiac muscle fate through the activation of Gata4/5/6 29 . The heart muscle will continue to differentiate and express myosin heavy chain 2 22 .
On the other hand, a subset of the TVCs express the transcription factor Tbx1/10, which promotes the expression of Ebf (COE, Collier/OLF/Ebf) and orchestrates the transition to the myogenic program by the activation of myogenic regulatory factor(Mrf), eventually giving rise to the longitudinal muscle and the muscles around the atrial siphon 29 . Another set of body muscle, the oral siphon muscles, derive from a different population of cells named trunk lateral cells (TLCs) in Ciona. TLCs follow a different fate but are partially regulated by the same transcription factors (TFs) involved in muscle development of TVCs 30,31 . After metamorphosis, all the body muscles continue to differentiate by expressing the same myosin heavy chain 3 22 .
In colonial species of ascidians, such as Botryllus schlosseri, the post-metamorphic individual, the oozooid, begins asexual budding from undifferentiated cells, in a process named blastogenesis, which leads to the development of an adult zooid, the blastozooid (Fig. 1). Therefore, the life cycle of a colonial ascidian is characterized by three different body plans: the larva displaying most chordate features, the post-metamorphic oozooid and the asexually propagated blastozooid. By producing multiple buds, in a cyclic manner, blastogenesis eventually leads to the formation of colonies composed of several genetically identical blastozooids. Regardless of their different ontogenetic origin, the overall anatomy of the oozooid and the blastozooid are similar, including the general organization of their musculature 20,24,32 . However, in blastogenesis the process of development is direct, namely it skips the determinative steps of embryonic development and passes neither through a larval stage nor metamorphosis 33 .
In order to investigate the mechanisms underlying clonal replication of muscular systems in colonial ascidians and infer the origin of muscle precursors during blastogenesis, we selected genes involved in embryonic-and-metamorphic myogenesis in the well-studied solitary ascidian Ciona, and followed their expression during blastogenesis of B. schlosseri. We took advantage of the extensive literature on myogenesis in solitary ascidians, as well as of the broad and detailed descriptions of Botryllus ontogeny 34 , to reconstruct the dynamics of muscle formation in ascidians.
Our study revealed that during myogenesis of an asexually derived zooid the embryonic and postmetamorphic myogenic genes are only partially co-opted, reflecting a lack of maternal signals and the absence of the larval stage.

Changes in muscle architecture during metamorphosis and blastogenesis
To understand whether the molecular structure of the differentiated muscles in colonial ascidians reflects the expression scenario described in solitary species 22 , and in order to compare the molecular structure of the muscle fibers between oozooid and blastozooid, we searched for members of the muscle type class two myosin heavy chains (MYH) in the transcriptomes of multiple stages in Botryllus schlosseri 35,36 . Four paralogues were identified in Botryllus schlosseri, including three muscle specific (MYH1, MYH2 and MYH3) and one non-muscle specific (MYH 9/10/11/14). The three muscle MYH clustered together with their corresponding homologous sequences of other solitary ascidians belonging to the suborder Phlebobranchia or Stolidobranchia (Supp. Fig.1). MYH3 subfamily constituted the sister group to the cluster of ascidian MYH1, MYH2, and their corresponding vertebrate paralogues. Within the ascidian paralogues, MYH1 and MYH2 6 clustered as the sister-group and were more closely related to the vertebrate muscle specific MYH sequences (Supp. Fig.1).
In situ hybridization revealed that the three mRNAs coding for the muscle relevant MYH proteins are expressed in different regions and at different time points of B. schlosseri development.
Embryonic Myh (Myh1) was exclusively expressed in the larval stage in tail muscles (Fig. 2 The heart in ascidians consists of a simple two-layered tube (myo-and pericard) that beats haemolymph in a reversible orientation through an open circulatory system 28,38 and is characterized by the expression of Myh2. The heart of solitary ascidians does not fully differentiate or begin contractions before metamorphosis, whereas the heart of some colonial ascidians may become fully functional before settlement 39 . In Botryllus, Myh2 expression was first observed at the ventral side of the swimming larva (Supp. Fig. 4.A1-5). The field displays already a sac-like form. In the early oozooid, Myh2 expression was observed on the beating heart at the left side of the body (Supp. Fig. 4.B1-5). In the fully developed oozooid heart, the muscle fibers of the heart, expressed Myh2 ( Fig. 2.C). Comparably, Myh2 expression in blastozooids was localized to the heart of primary buds, and expression was maintained throughout development and in the fully differentiated zooid (Fig. 2

Partial re-deployment of embryonic myogenic motifs during blastogenesis
To analyze if myogenic motifs expressed during embryonic development and metamorphoses have been co-opted during blastogenesis, we focused on the expression of 17 candidate genes well characterized during the myogenesis of solitary species 31,40 . We first described the embryonic development of the B. schlosseri to assay the conservancy of cleavage patterns between solitary and colonial species. We observed that the early cleavage patterns are comparable to the stereotypical patterns observed in solitary ascidians, including the extensively studied C. robusta 33,41,42 . However, the timing between each cleavage is longer, and the time from the first cleavage and the larval hatching is around 5 days (Supp. Fig. 5). The expression of Zic-r.a, Tbx6, Ebf and Tbx1/10 also confirmed the temporal and spatial pattern of expression observed in solitary species: the mRNA coding for the zinc finger transcription factor Zic-r.a was localized vegetally at the tip of the two blastomeres of the two-cell stage embryo (Supp. Fig. 6.A). At a 110-cell stage the expression localized in two posterior blastomeres (Supp. Fig. 6.B). Tbx6 mRNAs was expressed in a bilateral fashion (Supp. Fig. 6.C-D) in the presumptive myoplasm and future larval tail muscles (Supp. Fig. 6.E). Bilateral Ebf expression has been observed in a group of four single cells at the anterior trunk in an early tail bud stage (Supp. Fig. 6.F).
Next, we carried an initial assessment for presence and relative abundance of gene expression using the transcriptomes obtained from non-fertile colonies at seven blastogenetic stages 43 . These analyses showed the presence of only a subset of candidate genes, whereas others genes important for muscle development, typically expressed early in the embryonic development of Ciona were not found to be expressed during blastogenesis (Fig. 3). The absence of expression of early myogenic genes was also supported by RT-PCR (Supp. Fig. 6.G) and FISH (data not shown). The transcripts that were found in a negligibly low copy number or absent from the transcriptomes belong to myogenic transcription factors ZicL, LIM (Lhx3), Tbx6, Hand-r and Mesp, whereas Nk4, Tbx1/10 , FoxF, Islet, Myh2, Gata4/5/6, Mrf, Myh1 and Zic-r.a (Macho-1) were expressed at low levels. In contrast, Myh3, Ebf and Ets were myogenic factors expressed at high levels throughout the seven stages of Botryllus blastogenesis.

Development of the body wall musculature during Botryllus blastogenesis
In order to understand where the body wall muscles originate and develop during Botryllus blastogenesis, we investigated the expression of Tbx1/10, Ebf, and Mrf. These three TFs are expressed in one of the two muscle precursor fields in the Ciona larval head, specifically in the trunk ventral cells (TVCs). In contrast to descriptions during ascidian embryogenesis, we observed three main regions of expression of these genes in Botryllus blastogenesis: (1) a mesenchymal region between the dorsal tube and the dorsal epidermis, (2) the dorsal side of the branchial chamber where the future intersiphonal muscle will develop, and (3) the mantle or body wall, which corresponds to a membrane beneath the tunic made of epithelial tissue, connective tissue, musculature, blood vessels, and nerves ( Fig. 4.C,F,I).
Tbx1/10 expression began in the secondary bud at stage C2, in mesenchymal cells between the dorsal tube and the epidermis (Fig. 4.A). In the early primary bud (between stage D-A1), Tbx1/10 was expressed in mesenchymal cells distributed underneath the epidermis along the entire body of the zooid, i.e. the mantle (Fig. 4.D). At stage C2 the secondary bud started to express Tbx1/10 ventrally in the branchial chamber flanking the forming endostyle as well as in its medio-dorsal region ( Fig. 4.A-B). This second domain of expression appeared to concentrate laterally in the epithelium and into the cerebral ganglion region in stage D/A1 of the primary bud ( Fig. 4.E), and was maintained until A2, as it began to appear more diffuse. Tbx1/10 was also expressed on the ventral side of the bud within the region of the forming heart (see below).  (Fig. 4.H). In a C2 primary bud, Ebf was expressed in few scattered cells along the intersiphonal region (Fig. 4.J).
The transcriptomic data we analyzed showed a low expression of Mrf in all blastogenetic stages

Presence of putative muscle stem cells in the zooid
In Ciona, the atrial and oral siphon muscles have been shown to maintain a population of putative muscle stem cells that is Mrf-/bHLH-tun+ 31,44 . While Mrf is expressed in differentiating muscle cells, the tunicate-specific helix-loop-helix transcription factor bHLH-tun is expressed in muscle stem cells 31,44 . In the Botryllus oozooid, the bHLH-tun mRNA was expressed in a ring around the two siphons (Supp. Fig. 7.A1-5, B.1-4), which probably corresponds to the inner muscle population of the siphon, as was previously suggested in Ciona. In addition, bHLH-tun was expressed in a few cells of the lateral body wall (Supp. Fig. 7.A2) and in a few cells on top of the dorsal tube (Supp. Fig. 7.C1-4). In the Botryllus blastozooid, such spatial pattern of expression was not been detected, however transcriptome analyses showed that bHLH-tun is in fact present during different stages of blastogenesis (Fig. 3).

Heart development during blastogenesis
In order to investigate the formation of heart musculature during blastogenesis we followed the expression of the TFs FoxF ,Tbx1/10 and Nk4, which characterize the TVC embryonic lineage that gives rise to the heart muscles in Ciona 29,40 (Fig. 5). In contrast, Botryllus FoxF mRNA was expressed in cells of the bud epidermis throughout blastogenesis (Fig. 5.A-C), and more specifically on the dorsal side of the branchial chamber of the secondary bud on stage D (Fig. 5.A).
In the Botryllus primary bud, FoxF seemed to concentrate at sites lateral to the cerebral ganglion (data not shown). Last, we detected expression of FoxF in a cluster of cells in the left ventral side of the mantle where the heart formed ( Fig. 5.B), and in the heart of primary buds (Fig. 5.C).
Tbx1/10 transcripts showed similar patterns of expression to those described above for FoxF, but in addition Tbx1/10 showed expression on the secondary bud (stage D/A1) in a cluster of cells on the left ventral side of the mantle where the heart presumably will form (Fig. 5.D)., Heart field determination in Ciona is mainly characterized by the expression of Nk4, which antagonizes Tbx1/10 and Ebf 29 . In the Botryllus secondary bud at stage C2, Nk4 was expressed asymmetrically on the left side of the branchial chamber epithelium and in the entire left peribranchial chamber (Fig. 5.E). At stage B1, Nk4 expression was observed in the forming heart ( Fig. 5.F). Expression of Nk4 in the myocardium remained high during the complete development of the heart until the myocardium was separated from the pericardium (Fig. 5I).
To test whether Ebf and Nk4 expression domains are mutually exclusive, we performed double ISH (Fig. 5.G-H) on a single colony. Nk4 was expressed all over the secondary bud at stage B2-C1 except for the region of Ebf expression, which corresponds to the site where the dorsal tube will form (Fig. 5.G). We never found Ebf expressed at any stage of ascidian heart development ( Fig.   5.H).

DISCUSSION
In this study, we characterized mRNA expression patterns of myogenesis-related genes during the two different ontogeneses of the colonial ascidian Botryllus schlosseri. By following the sequential expression patterns of myogenic TFs throughout a complete series of developmental stages, we reconstructed the putative cellular precursors for body and heart muscles. We show that, within a single chordate species, the myogenic transcriptional motifs are only partially co-opted and cellular origin and transcriptional regulation that lead to adult muscles are coordinated differently during embryonic and non-embryonic developmental processes.

Colonial and solitary embryogenesis/metamorphosis share myogenic motifs
Comparative analyses of expression patterns of myogenic TFs during embryogenesis showed that several genes are conserved between Botryllus and the solitary species studied so far [45][46][47][48] suggesting that core elements of the myogenic regulatory cascade of solitary species are conserved during the sexual development of colonial species. For instance, the maternal expression of the posterior determinant and muscle specifier Zic-r.a (Macho-1) 45,49 in B. schlosseri, as well as its target Tbx6, indicate that the upstream determinants governing ascidian muscle development share the same expression as that reported for solitary forms. Ebf was also expressed during embryogenesis in B. schlosseri, and Mesp showed some expression despite the fact that it was not detectable by ISH, which we attribute to a temporal restriction of Mesp. Our data support the observation of Ricci et al. (2016) 33 showing that the early cleavage pattern seems to be conserved between solitary and colonial ascidians, at least for B. schlosseri. These data confirm the robustness of the ascidian expression and cleavage patterns across solitary and colonial species despite large variation in egg size, and strongly suggests that features of developmental mechanisms responsible of larval and post-metamorphic myogenesis might be well conserved in the whole class.

Differentiated muscle cells share molecular components in oozooids and blastozooids
In Botryllus, the body and heart muscles are formed anew, starting from the post-metamorphic oozooid and in every adult blastozooids during each blastogenetic cycle 20,24 . Regardless their different cellular origin and their divergent ontogenies, the oozooid and the blastozooid of Botryllus schlosseri present a similar arrangement of body structures, tissues, and cell types 32 . A few differences among the two zooid types, include differences in body size (the oozooid generally being bigger than the blastozooid), differences related to the architecture of the branchial basket 34,50 , and the organization of the musculature, e.g. the number of muscle fibers varies as well as their arrangement in the atrial siphon 24 . Despite the observed variation in muscle organization, both oozooid and blastozooid muscle cells expressed the same myosins in their fully differentiated fibers, i.e. Myh3 in the entire body wall musculature, and Myh2 in the heart. These results confirm that muscles of solitary and colonial ascidians express the same genes during final differentiation of muscle cells. Furthermore, within distinct zooids of the same colonial species, different developmental trajectories lead to a similar differentiation process and similar types of muscles.

Regulatory factors of myogenesis are only partially co-opted during blastogenesis
The transcriptomic profiles of the blastozooid developmental stages showed the presence of only a subset of the myogenic TFs engaged during ascidian embryogenesis. With the exception of Zic-r.a, upstream regulators of embryonic myogenesis, such as Tbx6, LIM (Lhx3), ZicL, Mesp, and Hand-r are not expressed in blastogenesis. During Botryllus embryogenesis, Zic-r.a is expressed in the neural plate, and during blastogenesis, it is expressed in the dorsal tube (Supp. Fig. 8). Zic-r.a expression in blastogenesis suggests a neurogenic rather than a myogenic function of Zic-r.a (Prünster et al, submitted). For instance, its expression does not seem linked to Tbx6, one of its downstream myogenic targets, which is absent in blastogenesis. This neurogenic role has been described in Ciona where, beside its function in early myogenesis, Zic-r.a is also zygotically expressed during neurogenesis, as suppressor of notochord fate 51 . The direct development of the bud, which lacks a larval stage with a notochord and tail musculature, may explain the absence of the early muscle transcriptional module. However, a lack of myogenic upstream regulators did not prevent the expression of late myogenic TFs, which are co-opted during blastogenesis. Precisely the same way that TFs are expressed in the TVCs of the Ciona larva throughout cardio-pharyngeal development 40 . These results suggests a degree of plasticity in the regulation of myogenic transcriptional modules in ascidians, which can be decoupled from the control of maternal determinants and early zygotic transcriptional regulators.

De novo origin of musculature in blastogenesis
In blastogenesis, the body muscle fate seems to be regulated by a kernel of genes that are expressed in the dorsal domain of the developing bud. In particular, Tbx1/10+ and Ebf+ cells are localized in a set of mesenchymal cells between the dorsal tube and the epidermis. These cells have been previously described in Botryllus and Diplosoma as neural precursors, which migrate and cluster forming the cerebral ganglion 52,53 . However, the sequential expression -both temporal and spatial-of Tbx1/10, Ebf and Mrf during bud development suggests that these cells migrate from the dorsal tube towards the lateral mantle, aligning where the future muscle fibers form There precursors might end up in the circular musculature of both siphons, as well as in the longitudinal muscle of the mantle. Therefore, in addition to center of neurogenesis, the dorsal tube could also have an additional role in myogenesis. In Ciona the oral siphon muscles do not derive from TVCs but from a population of TLCs, where Ebf is first expressed regulating downstream expression of Tbx1/10 30,31 . Without proper functional tests to dissect the interactions between Ebf and Tbx1/10, we cannot conclude whether the oral siphon musculature network of solitary ascidians is retained during blastogenesis. The presence of muscle stem cells cannot be completely ruled out due to the lack of detection of Mrf-/bHLH-tun+ cells by FISH in the blastozooids, because the colony transcriptomes revealed an expression of bHLH-tun. It remains to be illuminated whether a short-term, renewed every blastogenic cycle, or a long-lived population of muscle precursors persists, which simply remains undetectable by in situ hybridization so far.
Another domain of expression of Tbx1/10-Ebf-Mrf is located in a dorsal part of the branchial basket epithelium. In this domain, the temporal sequence of gene expression is different. Ebf is detected in the overhead mesenchymal cells during later developmental stages, and surprisingly Mrf is expressed before Ebf. The deviating patterns of expression of these genes as a result of heterochrony are suggestive of cell independent behaviors that occur at the site where the intersiphonal muscles will form. The intersiphonal muscles are the last muscles to form in the Botryllus blastozooid and connect longitudinally to the two siphons 24

During blastogenesis heart muscle origin is possibly uncoupled from body muscles
During blastogenesis, no evidence showed any morphological recapitulation of embryonic heart development, i.e. no ventral fusion of bilaterally located heart progenitors 28 . Instead, the heart either originate from mesenchymal precursors that cluster in the ventro-lateral side of the forming zooid 20,55 or arise in the same area from the evagination of the branchial chamber 56 . The heart lineage markers, Foxf, Nk4, Gata4/5/6 33 as well as TFs Tbx1/10 are expressed in the ventrolateral portion of the branchial chamber, where the cardiac muscle forms. Particularly the late Nk4 expression is restricted exclusively to the myocardium. While it remains difficult to live-track heart precursor cells and to find their origin, a correlation between the hierarchy of TFs expression and blastozooid organogenesis suggests that the heart is specified "in situ" in the ventro-lateral heart domain. The early expression of Tbx1/10 together with Nk4, that leads to Nk4 and Gata4/5/6 expression in the heart primordium resembles the formation of the second heart field 40 . However, the lack of functional relationships between TFs and the dramatically different ontogenesis suggests again a potential re-shuffling of the embryonic cardiac module.

CONCLUSIONS
In summary, the correlation between patterns of expression and morphogenesis presented here suggests the presence of three putative myogenic domains during Botryllus blastogenesis (Fig.   6.A): 1) muscle precursors delaminate from the dorsal tube and migrate along the mantle to form the circular muscles of the siphons and body wall muscles; 2) intersiphonal muscles originate from a dorsal portion of the branchial chamber and seem to be regulated in a different way from the other body wall muscles; and 3) the heart is formed from another population of founder cells localized in a ventro-lateral domain of the bud, which remains poorly characterized.
The origin of muscle cells in B. schlosseri blastogenesis is elusive but specifier of cardiac and adult body wall muscles appear partially coopted (Fig 6. B).

Animal husbandry
Botryllus schlosseri colonies were raised on a 50x70x1 mm glass slides as described previously 61 .
A Botryllus colony consists of three coexisting asexual generations: the adult filtering zooids, their buds, called primary buds, and the secondary buds (or budlets), sprouting from the primary buds ( Fig. 1). Budding (blastogenesis) was staged according to Lauzon et al. (2002) 62 . First a budlet appears as thickening of the peribranchial chamber and overlying epidermis of the adult zooid (stage A), thus closes off forming a double vesicle connected with the parental zooid by the epidermis (stage B). Then organogenesis begins and the inner vesicle separates into three chambers: one central branchial and two lateral peribranchial chambers (stage C). Staging of the development of the embryo has been done according to Conklin 63 .

Embryo harvesting and dechorionation
Embryos were harvested at different developmental stages from the colony by opening the Botryllus adults with a syringe. Dechorionation was performed in fertilized eggs by shaking the eggs at 60RPM at room temperature in 0.2 % trypsin and 20 mM TAPS, pH 8.2 in seawater for 1.5-2 hours followed by several seawater washes. To calculate the cleavage time, nondechorionated fertilized eggs have been harvested and kept in filtered seawater at 17°C.

Fluorescent in situ hybridization (FISH)
Primers for antisense mRNA probes were designed in the translated region of each gene (Supp. Table 1) FISH was carried out as previously described in Ricci et al. (2016) with the following modifications: 1% Dextran sulfate was added to the Hybridization buffer and the revelation solution. The anti-Digoxigenin Antibody (HRP) (Roche,11207733910) was pre-adsorbed for 1 hour in hybridization solution with a mix of fixed colonies at different stages. When the tunic was exhibiting a very strong background, the animals were manually removed from x the tunic after rehydration, post fixed in 4% PFA for 1h and transferred into washing baskets in 24-well plates.
DIG-probe detection was performed with bench-made FITC-Tyramide by 3h incubation. For double FISH, the hybridization of DIG labeled and Fluorescein labeled probes was performed at the same time, fluorescein probes where detected with Cy3-Tyramide. The ISH on embryos was performed after   65 .

Transcriptional data analysis
Transcripts of RNA-seq data of seven stages of an unfertile colony of B. schlosseri SB802d 43 were quantified by pseudo alignment via Kallisto 66 to the mixed stage transcriptome database http://octopus.obs-fr.fr/public/botryllus/blast_botryllus.php. Heat map was generated using R, a cutoff for not expressed genes was chosen <1 RPM, lowly expressed 1-50, and high expressed >50 RPM. Orthologues of muscle determinants have been selected by reciprocal blast with published data from NCBI. For some candidate genes, the orthology has been also assessed by phylogenetic analyses (Supp. Fig. 10-14, Supp. Text 1).

Imaging
Imaging of the NBT/BCIP ISH have been acquired with Zeiss Axio Imager A2 with a 20x magnification, DIC, and color camera. Confocal images were acquired a Leica SP8 (40x/1.1 Water WD 0.6 HCX PL APO CS2) and processed with ImageJ and Inkscape.

Fig. 1. Development and staging of a Botryllus schlosseri colony.
The staging of the animals was performed after Lauzon 67 . The secondary bud develops as thickening of the peribranchial epithelium and the epidermis (stages A1, A2), which evaginates and closes forming a double monolayered vesicle (stages B1, B2). The inner vesicle undergoes morphogenesis and is subdivided into three chambers (stages C1, C2). During "takeover" (stages), the adult degenerate and get resorbed, the primary bud become adult, the secondary bud becomes the primary bud and a new blastogenetic cycle begins for the next secondary bud.    For what concern Mef2 expression no information are currently available in ascidians 72 . An orthologue to vertebrate Mef2, is present in the transcriptomic dataset of blastogenesis.
Pharyngeal muscle start differentiation by activating the paralogues Myf5 and MyoD; only one orthologue, Mrf, is found in ascidians. To activate such in ascidians Ebf is expressed in the body muscle lineage. Two myosin heavy chain forms characterize the vertebrate heart. Pharyngeal muscles do not express the same isoforms in all vertebrates: ruminants and rodents express the same myosins as in trunk and limb, namely Myh1 & 2, in other animals Myh 6 and Myh16 can be expressed in addition. In ascidians, Myh2 is expressed in the heart, and Myh3 in the body wall, their proteins are not orthologues of vertebrate Myh2 and 3 (Supp. Fig. 1.). Bolt text indicates conserved expression within at least two species, gray only transcriptomic data, asterisk only in some species.