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Cardiopharyngeal deconstruction and ancestral tunicate sessility

Nature volume 599, pages 431–435 (2021)Cite this article

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Abstract

A central question in chordate evolution is the origin of sessility in adult ascidians, and whether the appendicularian complete free-living style represents a primitive or derived condition among tunicates1. According to the ‘a new heart for a new head’ hypothesis, the evolution of the cardiopharyngeal gene regulatory network appears as a pivotal aspect to understand the evolution of the lifestyles of chordates2,3,4. Here we show that appendicularians experienced massive ancestral losses of cardiopharyngeal genes and subfunctions, leading to the ‘deconstruction’ of two ancestral modules of the tunicate cardiopharyngeal gene regulatory network. In ascidians, these modules are related to early and late multipotency, which is involved in lineage cell-fate determination towards the first and second heart fields and siphon muscles. Our work shows that the deconstruction of the cardiopharyngeal gene regulatory network involved the regressive loss of the siphon muscle, supporting an evolutionary scenario in which ancestral tunicates had a sessile ascidian-like adult lifestyle. In agreement with this scenario, our findings also suggest that this deconstruction contributed to the acceleration of cardiogenesis and the redesign of the heart into an open-wide laminar structure in appendicularians as evolutionary adaptations during their transition to a complete pelagic free-living style upon the innovation of the food-filtering house5.

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Fig. 1: O. dioica cardiogenesis.
Fig. 2: Comparison of the cardiopharyngeal cell lineage and GRN in ascidians and appendicularians.
Fig. 3: Evolutionary scenario of the deconstruction of the cardiopharyngeal GRN and acquisition of an adult free-living style in appendicularians.

Data availability

Accession numbers and URLs of databases from publicly available sources are provided in the Methods, Supplementary Information and Supplementary Data 1.

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Acknowledgements

We thank all team members of the C.C. and R.A. laboratories for discussions; S. Artime for assistance with the animal facility; and L. Christiaen and A. Elewa for reading and commenting on the manuscript. C.C. was supported by BFU2016-80601-P and PID2019-110562GB-I00, R.A. by BIO2015-67358-C2-1-P and J.G.-F. by BFU2017-861152-P and PID2020-117820GB-I00 grants from Ministerio de Ciencia y Innovación (Spain). C.C., R.A. and J.G.-F. were also supported by grant 2017-SGR-1665 from Generalitat de Catalunya. A.F.-R. was supported by FPU14/02654, G.S.-S. by FPU18/02414, M.P.-C. by colaboración-2015/16, P.B. by colaboración-2016/17 and M.J.-L. by colaboración-2019/20 fellowships from Ministerio de Educación Cultura y Deporte. M.F.-T. was supported by a PREDOC2020/58 fellowship from the University of Barcelona. M.P.-C. was supported by PPL1415, A.F.-R by PPL1314 and P.B. by PPLB1617 from Asociación Española Contra el Cáncer (AECC).

Author information

Author notes

  1. These authors contributed equally: Marc Fabregà-Torrus and Gaspar Sánchez-Serna

Authors and Affiliations

  1. Departament de Genètica, Microbiologia i Estadística, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

    Alfonso Ferrández-Roldán, Marc Fabregà-Torrus, Gaspar Sánchez-Serna, Enya Duran-Bello, Martí Joaquín-Lluís, Paula Bujosa, Marcos Plana-Carmona, Jordi Garcia-Fernàndez, Ricard Albalat & Cristian Cañestro

  2. Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Barcelona, Spain

    Alfonso Ferrández-Roldán, Marc Fabregà-Torrus, Gaspar Sánchez-Serna, Enya Duran-Bello, Martí Joaquín-Lluís, Paula Bujosa, Marcos Plana-Carmona, Ricard Albalat & Cristian Cañestro

  3. Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain

    Jordi Garcia-Fernàndez

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Contributions

A.F.-R. carried out the cardiac developmental atlas, genome surveys, phylogenetic analyses, WMISH experiments, cell lineage mapping and BMP inhibitory treatments. M.F.-T. contributed to the Gata genome survey and FWMISH. G.S.-S. and P.B. contributed to the Fgf/Mapk genome survey and FGF inhibitory treatments. E.D.-B. contributed to the Tbx genome survey and WMISH. M.J.-L. contributed to the Ets and Tbx phylogenies. M.P.-C. contributed to Islet characterization. A.F.-R. interpreted the data and made the figures. C.C. conceptualized the project. J.G.-F. and R.A. provided resources. R.A. and C.C. supervised the experiments. C.C. and A.F.-R. wrote the manuscript. All authors commented on the manuscript and agreed to its final version.

Corresponding author

Correspondence to Cristian Cañestro.

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The authors declare no competing interests.

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Peer review information Nature thanks Benoit Bruneau, Brad Davidson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 4D-reconstruction of a virtual cardiac cell tracing, based on nuclear position from the 30-cell stage to tailbud stages of O. dioica embryos (modified from Stach 2008)16.

B8.9 appears as the first CPC. Blastomere nomenclature follows that of Conklin for ascidians (vegetal blastomeres in capital letters, animal blastomeres in small letters, and blastomeres from the right underlined)38, and their fate are indicated in different colors: muscle+heart (purple), posterior tail muscle cells (yellow), anterior tail muscle cells (ATM, green), heart (red), germ-line (blue). Circles and hexagons represent blastomeres derived from right and left sides of the embryo, respectively. Dashes encircle sister cells resulting from a cell division.

Extended Data Fig. 2 Mesp ML phylogenetic tree and Math and Neurogenin expression.

a, Unrooted phylogenetic tree, represented in a rectangular layout for the sake of clarity, showing the presence of bHLH homologs of Neurogenin and Math in appendicularians, but the absence of Mesp. The presence of Mesp in cephalochordates, vertebrates and all analyzed ascidians suggests an ancestral loss of Mesp at the base of the appendicularian lineage after its split from the lineage leading to ascidians. Bootstrap values are shown in the nodes. Scale bar indicates amino acid substitutions. Vertebrates (black): Gallus gallus (Gga), Homo sapiens (Hsa), Latimeria chalumnae (Lch), Lepisosteus oculatus (Loc); Ascidian tunicates (blue): Botrylloides leachii (Ble), Botrylloides schlosseri (Bsc), Ciona robusta (Cro), Ciona savignyi (Csa), Halocynthia aurantium (Hau), Halocynthia roretzi (Hro), Molgula occidentalis (Mocci), Molgula occulta (Moccu), Molgula oculata (Mocul), Phallusia fumigata (Pfu), Phallusia mammillata (Pma); Appendicularian tunicates (red): Bathochordaeus sp. (Bsp), Fritillaria borealis (Fbo), Mesochordaeus erythrocephalus (Mer), Oikopleura albicans (Oal), Oikopleura dioica (Odi), Oikopleura longicauda (Olo), Oikopleura vanhoeffeni (Ova); Cephalochordates (green): Branchiostoma belcheri (Bbe), Branchiostoma floridae (Bfl), Branchiostoma lanceolatum (Bla). b–f, Developmental expression pattern of O. dioica Math homolog. Whole mount in situ hybridization in different stages of O. dioica development showing expression in the notochord in tailbud and early-hatchling embryos (red arrowheads) (c, d), in epidermis (blue arrowheads) (c–f), in the rectum domain in hatchling stages (yellow arrowheads) (d–f), in later stages of neural system development (pink arrowheads) (e, f), and in later stages of digestive system development (green arrowheads) (e, f). g–k, Developmental expression pattern of O. dioica Neurogenin homolog. Whole mount in situ hybridization in different stages of O. dioica development shows that Neurogenin expression was restricted to nervous system in tailbud and early-hatchling stages (pink arrowheads) (h, i) but no expression was detected in any region compatible with cardiac function. Images from tailbud in advance correspond to left lateral views orientated anterior towards the left and dorsal towards the top.

Extended Data Fig. 3 Ets ML phylogenetic tree and expression.

a, Unrooted phylogenetic tree of the Ets and Erg protein families showed a high bootstrap value separating both protein families what corroborated the existence of two Ets1/2 genes in appendicularians. Scale bar indicates amino acid substitutions. Vertebrates (black): Gallus gallus (Gga), Homo sapiens (Hsa), Latimeria chalumnae (Lch), Lepisosteus oculatus (Loc); Ascidian tunicates (blue): Botrylloides leachii (Ble), Botrylloides schlosseri (Bsc), Ciona robusta (Cro), Ciona savignyi (Csa), Halocynthia aurantium (Hau), Halocynthia roretzi (Hro), Molgula occidentalis (Mocci), Molgula occulta (Moccu), Molgula oculata (Mocul), Phallusia fumigata (Pfu), Phallusia mammillata (Pma); Appendicularian tunicates (red): Bathochordaeus sp. (Bsp), Fritillaria borealis (Fbo), Mesochordaeus erythrocephalus (Mer), Oikopleura albicans (Oal), Oikopleura dioica (Odi), Oikopleura longicauda (Olo), Oikopleura vanhoeffeni (Ova); Cephalochordates (green): Branchiostoma belcheri (Bbe), Branchiostoma floridae (Bfl), Branchiostoma lanceolatum (Bla). b, Phylogenetic analysis of chordate Ets1/2, using cephalochordate sequences as outgroup, suggested that the two Ets1/2 genes of appendicularians were co-orthologs to the ascidian Ets1/2a. c–h, Whole mount in situ hybridization of O. dioica Ets1/2a1 did not show any clear expression before hatchling stages (c–f). In early-hatchling stage Ets1/2a1 revealed expression in the migratory endodermal strand cells (pink arrowheads) (g). In late-hatchling the expression signal was restricted to the buccal gland (green arrowheads) (h). i, j, Ets1/2a2 did not show expression until tailbud stage. k, l, In tailbud embryos, expression signal was detected in tail muscle cells (orange arrowheads), the notochord (red arrowheads) and the epidermis of the trunk (blue arrowheads). m, In early-hatchling expression signal continued in the tail muscle and the notochord and increased in the anal domain (yellow arrowhead). n, In late-hatchling stage, the Ets1/2a2 expression covered the entire oikoplastic epithelium, and continued in the muscle cells of the tail. Large images from tailbud in advance correspond to left lateral views oriented anterior towards the left and dorsal towards the top. Inset images are dorsal views of optical cross sections at the levels of dashed lines.

Extended Data Fig. 4 FGF/MAPK ML phylogenetic tree and expression.

a, ML phylogenetic tree of the MEK subfamilies in chordates revealing the loss of the MEK4, MEK5 and MEK1/2 subfamilies in appendicularians, but the surviving of MEK3/6 and MEK7 subfamilies. Scale bar indicates amino acid substitutions. Bootstrap values are shown in the nodes. Vertebrates (black): Gallus gallus (Gga), Homo sapiens (Hsa), Latimeria chalumnae (Lch), Lepisosteus oculatus (Loc); Ascidian tunicates (blue): Botrylloides leachii (Ble), Botrylloides schlosseri (Bsc), Ciona robusta (Cro), Ciona savignyi (Csa), Halocynthia aurantium (Hau), Halocynthia roretzi (Hro), Molgula occidentalis (Mocci), Molgula occulta (Moccu), Molgula oculata (Mocul), Phallusia fumigata (Pfu), Phallusia mammillata (Pma); Appendicularian tunicates (red): Bathochordaeus sp. (Bsp), Fritillaria borealis (Fbo), Mesochordaeus erythrocephalus (Mer), Oikopleura albicans (Oal), Oikopleura dioica (Odi), Oikopleura longicauda (Olo), Oikopleura vanhoeffeni (Ova); Cephalochordates (green): Branchiostoma belcheri (Bbe), Branchiostoma floridae (Bfl), Branchiostoma lanceolatum (Bla). b–g, Whole mount in situ hybridization of ERK homolog in different stages of O. dioica development did not detect expression in any studied stage (b–f) until late-hatchling when expression was detected in an specific central domain in the oikoplastic epithelium (blue arrowheads) (g). h–m, Whole mount in situ hybridization of MEK7 homolog in O. dioica revealed expression in the developing neural tissue in tailbud stages (pink arrowheads) (i, j), and in the esophagus (green arrowhead) and the oikoplastic epithelium (blue arrowheads) in the late-hatchling stage (m). n–s, Whole mount in situ hybridization of MEK3/6 homolog in different stages of O. dioica development did not show any obvious tissue specific expression domain in the trunk, but the signal was generalized, with the exception of muscle cells in the tail at late-hatchling stages. Images from tailbud in advanced correspond to left lateral views orientated anterior towards the left and dorsal towards the top.

Extended Data Fig. 5 Gata and FoxF ML phylogenetic trees in chordates.

a, Gata ML phylogenetic tree reveals the loss of the Gata4/5/6 in appendicularians, but the surviving and lineage specific duplications of Gata1/2/3 in appendicularians. b, FoxF ML phylogenetic tree reveals the presence of an ortholog of FoxF in appendicularians. The sister FoxQ subfamily was used as outgroup to root the tree. Scale bar indicates amino acid substitutions. Bootstrap values are shown in the nodes. Vertebrates (black): Gallus gallus (Gga), Homo sapiens (Hsa), Latimeria chalumnae (Lch), Lepisosteus oculatus (Loc); Ascidian tunicates (blue): Botrylloides leachii (Ble), Botrylloides schlosseri (Bsc), Ciona robusta (Cro), Ciona savignyi (Csa), Halocynthia aurantium (Hau), Halocynthia roretzi (Hro), Molgula occidentalis (Mocci), Molgula occulta (Moccu), Molgula oculata (Mocul), Phallusia fumigata (Pfu), Phallusia mammillata (Pma); Appendicularian tunicates (red): Bathochordaeus sp. (Bsp), Fritillaria borealis (Fbo), Mesochordaeus erythrocephalus (Mer), Oikopleura albicans (Oal), Oikopleura dioica (Odi), Oikopleura longicauda (Olo), Oikopleura vanhoeffeni (Ova); Cephalochordates (Green): Branchiostoma belcheri (Bbe), Branchiostoma floridae (Bfl), Branchiostoma lanceolatum (Bla).

Extended Data Fig. 6 NK ML phylogenetic tree in chordates reveals the presence of an ortholog of Nk4 in appendicularians and two orthologs of the Nk2 subfamily.

Scale bar indicates amino acid substitutions. Bootstrap values are shown in the nodes. Vertebrates (black): Gallus gallus (Gga), Homo sapiens (Hsa), Latimeria chalumnae (Lch), Lepisosteus oculatus (Loc); Ascidian tunicates (blue): Botrylloides leachii (Ble), Botrylloides schlosseri (Bsc), Ciona robusta (Cro), Ciona savignyi (Csa), Halocynthia aurantium (Hau), Halocynthia roretzi (Hro), Molgula occidentalis (Mocci), Molgula occulta (Moccu), Molgula oculata (Mocul), Phallusia fumigata (Pfu), Phallusia mammillata (Pma); Appendicularian tunicates (red): Bathochordaeus sp. (Bsp), Fritillaria borealis (Fbo), Mesochordaeus erythrocephalus (Mer), Oikopleura albicans (Oal), Oikopleura dioica (Odi), Oikopleura longicauda (Olo), Oikopleura vanhoeffeni (Ova); Cephalochordates (Green): Branchiostoma belcheri (Bbe), Branchiostoma floridae (Bfl), Branchiostoma lanceolatum (Bla).

Extended Data Fig. 7 Hand ML phylogenetic tree suggests that member of this family in O. dioica is homologous to ascidian Hand1/2.

Despite the tree suggests that the second paralog of ascidian (Hand-r) arose by a duplication at the base of the tunicate clade, and therefore subsequently lost in appendicularians. The low node support –bootstrap and approximate likelihood-ratio test (aLRT)– and the presence of shared long amino acid domain rich in K between the Hand1/2 and Hand-r in ascidians, but absent in appendicularians, do not allow us to discard the possibility that Hand-r was originated by a duplication within the ascidian lineage, and its basal branching in the tunicate clade is due to a long branch attraction phenomenon. Scale bar indicates amino acid substitutions. Bootstrap values are shown in the nodes. Vertebrates (black): Gallus gallus (Gga), Homo sapiens (Hsa), Latimeria chalumnae (Lch), Lepisosteus oculatus (Loc); Ascidian tunicates (blue): Botrylloides leachii (Ble), Botrylloides schlosseri (Bsc), Ciona robusta (Cro), Ciona savignyi (Csa), Halocynthia aurantium (Hau), Halocynthia roretzi (Hro), Molgula occidentalis (Mocci), Molgula occulta (Moccu), Molgula oculata (Mocul), Phallusia fumigata (Pfu), Phallusia mammillata (Pma); Appendicularian tunicates (red): Bathochordaeus sp. (Bsp), Fritillaria borealis (Fbo), Mesochordaeus erythrocephalus (Mer), Oikopleura albicans (Oal), Oikopleura dioica (Odi), Oikopleura longicauda (Olo), Oikopleura vanhoeffeni (Ova); Cephalochordates (green): Branchiostoma belcheri (Bbe), Branchiostoma floridae (Bfl), Branchiostoma lanceolatum (Bla).

Extended Data Fig. 8 Developmental coexpression patterns of ActnM1 and potential cardiac transcription factors.

Double fluorescent in situ hybridization of ActnM1 with Nk4, Hand1/2, FoxF, Gata1/2/3b and Gata1/2/3d. Nk4 expression signal was detected in ventral epidermis and the CPC (B8.9) from the incipient-tailbud stage (a) until the early-tailbud (a’). In later stages, we only detected expression in the epidermis, but not in the cardiac precursors (a’’–a’’’). Hand1/2 was specifically expressed in the cardiac progenitors from late-tailbud to hatchling stages (b’’–b’’’). We did not detect expression of FoxF, Gata1/2/3b nor Gata1/2/3d in cardiac precursors, but they were expressed in different epidermal domains (c–e’’’). The images correspond to the overlay of a stack of confocal sections with expression of the different genes. The small overlapping color in e’’’ is due to the overlay of the stack, and not to actual co-expression. White arrowheads indicate co-expression of ActnM1 with the corresponding gene in cardiac progenitors. Incipient- and early-tailbud stages correspond to ventral views oriented anterior towards the top. Late-tailbud and early-hatchling stages correspond to lateral views oriented anterior towards the left and dorsal towards the top.

Extended Data Fig. 9 FGF, MEK and BMP inhibition during heart development of O. dioica.

a–g’, Whole mount in situ hybridization of ActnM1 in DMSO-control (a) and treated embryos with inhibitors of FGFR (SU5402 and AZD4547), MEK3/6 (Gossypetin), MEK7 (5Z-7-Oxozeaenol) and BMP inhibitors (LDN and Dorsomorphin) from 2-cell stage up to early-tailbud stage (b–g’). Embryos treated with FGFR and MEK inhibitors affected gastrulation and caused abnormal phenotypes in which mesodermal derivatives showed either abnormal domains (b’–e’) or complete absence (b’’–e’’). However, those treated embryos that reached fairly normal incipient morphologies (b–g), showed the presence of CPCs (red arrowheads). h–n’, Whole mount in situ hybridization of NK4+Brachyury in DMSO-control (h) and treated embryos with FGFR, MEK and BMP inhibitors (i–n’) from 32-cell stage to early-tailbud stage. A majority of the treated embryos showed the Nk4 expression in the CPCs (i–n), even in some with obvious abnormalities in the notochord (i). Only in embryos with severe abnormal morphologies or arrested, we could not distinguish the CPCs from other Nk4 expression domains (i’–n’). o–t’, Whole mount in situ hybridization of ActnM1 in DMSO-control (o) and treated embryos with FGFR, MEK and BMP inhibitors from 32-cell stage to early-hatchling stage (p–t’). Most of the treated embryos showed abnormal tails (p’–t’), in which the elongation and rotation had been affected. Moreover, while the CPCs had converged near the midline into a single cardiac field, we observed that in many embryos with tail malformations, the CPCs had not converged and were still bilaterally separated at the right and left sides of the trunk (red numbers in brackets). These results suggests that FGF/MEK/MAPK and BMP signaling pathways may be involved in tail elongation/rotation and late cardiac organogenesis. Tailbud embryos images correspond to dorsal views with anterior to the left. Hatchling images represent dorsal views with anterior to the top.

Extended Data Fig. 10 Tbx ML phylogenetic tree and Islet, Ebf, MyoD and Dach expression.

a, ML phylogenetic tree of the Tbx subfamilies in chordates reveals the loss of Tbx1/10 and Tbx21/Eomes/Tbr1 subfamilies in appendicularians and the ancestral loss of Tbx4/5 subfamily in tunicates. Scale bar indicates amino acid substitutions. Bootstrap values are shown in the nodes. Vertebrates (black): Gallus gallus (Gga), Homo sapiens (Hsa), Latimeria chalumnae (Lch), Lepisosteus oculatus (Loc); Ascidian tunicates (blue): Botrylloides leachii (Ble), Botrylloides schlosseri (Bsc), Ciona robusta (Cro), Ciona savignyi (Csa), Halocynthia aurantium (Hau), Halocynthia roretzi (Hro), Molgula occidentalis (Moocci), Molgula occulta (Mooccu), Molgula oculata (Moocul), Phallusia fumigata (Pfu), Phallusia mammillata (Pma); Ascidian appendicularians (red): Bathochordaeus sp. (Bsp), Fritillaria borealis (Fbo), Mesochordaeus erythrocephalus (Mer), Oikopleura albicans (Oal), Oikopleura dioica (Odi), Oikopleura longicauda (Olo), Oikopleura vanhoeffeni (Ova); Cephalochordates (green): Branchiostoma floridae (Bfl), Branchiostoma lanceolatum (Bla). b–w, Whole mount in situ hybridization of O. dioica Islet, Ebf, MyoD and Dach homologs. 64-cell embryos did not showed expression of Islet (b) which was only detected in the developing nervous system from tailbud to hatchling embryos (c–f). Ebf (COE) did not show expression in early stages (g, h) but we detected expression in the nervous system from tailbud to mid-hatchling stage (i–k) and in the oikoplastic epithelium of late-hatchling embryos (l). We did not detect expression of MyoD from 32-cell to hatchling embryos (m–p). In late-hatchling embryos MyoD was expressed in the oikoplastic epithelium (q). Dach expression started at the 64-cell stage in the developing nervous system (pink arrowheads) and continued until late-tailbud stage (r–t). In tailbud stages, Dach started expressing in the trunk epidermis (blue arrowheads) which was maintained until late-hatchling stages when it was expressed in the whole oikoplastic epithelium (blue arrowheads) (s–v). In mid-hatchling stage, beside the epidermis, Dach expression was also detected in the endostyle (green arrowheads) (w). Large images from tailbud in advance correspond to left lateral views oriented anterior towards the left and dorsal towards the top. Inset images are dorsal views of optical cross sections at the levels of dashed lines. Pink arrowheads indicate the developing nervous system. Blue arrowheads indicate the oikoplastic epithelium.

Supplementary information

Supplementary Information

This file contains the Supplementary Discussion and Supplementary References.

Reporting Summary

Supplementary Video 1

Ventral view of a late hatchling at 8.5 hours post fertilization showing the first heart beatings, before the rest of the organs of the trunk are fully developed.

Supplementary Data 1

Accession numbers and species abbreviations.

Supplementary Data 2

FGF/MEK and BMP inhibitory treatment results.

Supplementary Data 3

Cardiac cell-specific marker BLAST identification

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Ferrández-Roldán, A., Fabregà-Torrus, M., Sánchez-Serna, G. et al. Cardiopharyngeal deconstruction and ancestral tunicate sessility. Nature 599, 431–435 (2021). https://doi.org/10.1038/s41586-021-04041-w

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