Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

Center for Developmental Genetics Department of Biology New York University New York NY 10003 USA

Department of Anatomy and Cell Biology Kansas University Medical Center Kansas City KS 66160 USA

Division of Biology and Biological Engineering California Institute of Technology Pasadena CA 91125 USA

Friedrich Miescher Laboratory of the Max Planck Society Tübingen 72076 Germany

Institute of Molecular Genetics of the ASCR Prague 142 20 Czech Republic

Institute of Molecular Life Sciences University of Zurich Zürich 8057 Switzerland

Max Planck Institute of Molecular Cell Biology and Genetics Dresden 01307 Germany

Morgridge Institute for Research Madison WI 53715 USA

Stowers Institute for Medical Research Kansas City MO 64110 USA

TUD CRTD Center for Regenerative Therapies Dresden Dresden 01307 Germany

Zobrazit více v PubMed

Gurdon, J. B. Organization of the Early Vertebrate Embryo 51–59 (Springer, Boston, MA US, 1995).

Takasato M, Little MH. The origin of the mammalian kidney: implications for recreating the kidney in vitro. Development. 2015;142:1937–1947. doi: 10.1242/dev.104802. PubMed DOI

Chal J, Pourquié O. Making muscle: skeletal myogenesis in vivo and in vitro. Development. 2017;144:2104–2122. doi: 10.1242/dev.151035. PubMed DOI

Lane MC, Smith WC. The origins of primitive blood in Xenopus: implications for axial patterning. Development. 1999;126:423–434. PubMed

Davidson AJ, Zon LI. The ‘definitive’ (and ‘primitive’) guide to zebrafish hematopoiesis. Oncogene. 2004;23:7233–7246. doi: 10.1038/sj.onc.1207943. PubMed DOI

Yabe T, Hoshijima K, Yamamoto T, Takada S. Quadruple zebrafish mutant reveals different roles of Mesp genes in somite segmentation between mouse and zebrafish. Development. 2016;143:2842–2852. doi: 10.1242/dev.133173. PubMed DOI PMC

Kusakabe R, Kuratani S. Evolutionary perspectives from development of mesodermal components in the lamprey. Dev. Dyn. 2007;236:2410–2420. doi: 10.1002/dvdy.21177. PubMed DOI

Pascual-Anaya J, et al. The evolutionary origins of chordate hematopoiesis and vertebrate endothelia. Dev. Biol. 2013;375:182–192. doi: 10.1016/j.ydbio.2012.11.015. PubMed DOI

Onimaru K, Shoguchi E, Kuratani S, Tanaka M. Development and evolution of the lateral plate mesoderm: comparative analysis of amphioxus and lamprey with implications for the acquisition of paired fins. Dev. Biol. 2011;359:124–136. doi: 10.1016/j.ydbio.2011.08.003. PubMed DOI

Kaplan N, Razy-Krajka F, Christiaen L. Regulation and evolution of cardiopharyngeal cell identity and behavior: insights from simple chordates. Curr. Opin. Genet. Dev. 2015;32:119–128. doi: 10.1016/j.gde.2015.02.008. PubMed DOI PMC

Becker D, Eid R, Schughart K. The limb/LPM enhancer of the murine Hoxb6 gene: reporter gene analysis in transgenic embryos and studies of DNA-protein interactions. Pharm. Acta Helv. 1996;71:29–35. doi: 10.1016/0031-6865(95)00049-6. PubMed DOI

Rojas A, et al. Gata4 expression in lateral mesoderm is downstream of BMP4 and is activated directly by Forkhead and GATA transcription factors through a distal enhancer element. Development. 2005;132:3405–3417. doi: 10.1242/dev.01913. PubMed DOI

Chandler KJ, Chandler RL, Mortlock DP. Identification of an ancient Bmp4 mesoderm enhancer located 46 kb from the promoter. Dev. Biol. 2009;327:590–602. doi: 10.1016/j.ydbio.2008.12.033. PubMed DOI PMC

Mosimann C, et al. Chamber identity programs drive early functional partitioning of the heart. Nat. Commun. 2015;6:8146. doi: 10.1038/ncomms9146. PubMed DOI PMC

Gays D, et al. An exclusive cellular and molecular network governs intestinal smooth muscle cell differentiation in vertebrates. Development. 2017;144:464–478. doi: 10.1242/dev.133926. PubMed DOI

Felker A, et al. Continuous addition of progenitors forms the cardiac ventricle in zebrafish. Nat. Commun. 2018;9:2001. doi: 10.1038/s41467-018-04402-6. PubMed DOI PMC

Pimtong W, Datta M, Ulrich AM, Rhodes J, Zon LI. Drl.3 governs primitive hematopoiesis in zebrafish. Sci. Rep. 2015;4:5791. doi: 10.1038/srep05791. PubMed DOI PMC

Sumanas S, Zhang B, Dai R, Lin S. 15-Zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation. Dev. Biol. 2005;283:85–96. doi: 10.1016/j.ydbio.2005.04.007. PubMed DOI

Alexander J, Rothenberg M, Henry GL, Stainier DY. Casanova plays an early and essential role in endoderm formation in zebrafish. Dev. Biol. 1999;215:343–357. doi: 10.1006/dbio.1999.9441. PubMed DOI

Herbomel P, Thisse B, Thisse C. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development. 1999;126:3735–3745. PubMed

Emerson RO, Thomas JH. Adaptive evolution in zinc finger transcription factors. PLoS Genet. 2009;5:e1000325. doi: 10.1371/journal.pgen.1000325. PubMed DOI PMC

Kaufman CK, et al. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science. 2016;351:aad2197. doi: 10.1126/science.aad2197. PubMed DOI PMC

Langdon YG, Mullins MC. Maternal and zygotic control of zebrafish dorsoventral axial patterning. Annu Rev. Genet. 2011;45:357–377. doi: 10.1146/annurev-genet-110410-132517. PubMed DOI

Hild M, et al. The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo. Development. 1999;126:2149–2159. PubMed

Gritsman K, et al. The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell. 1999;97:121–132. doi: 10.1016/S0092-8674(00)80720-5. PubMed DOI

Nelson AC, et al. In vivo regulation of the zebrafish endoderm progenitor niche by T-Box transcription factors. Cell Rep. 2017;19:2782–2795. doi: 10.1016/j.celrep.2017.06.011. PubMed DOI PMC

Nelson AC, et al. Global identification of Smad2 and Eomesodermin targets in zebrafish identifies a conserved transcriptional network in mesendoderm and a novel role for Eomesodermin in repression of ectodermal gene expression. BMC Biol. 2014;12:81. doi: 10.1186/s12915-014-0081-5. PubMed DOI PMC

Dubrulle J, et al. Response to Nodal morphogen gradient is determined by the kinetics of target gene induction. Elife. 2015;4:e05042. doi: 10.7554/eLife.05042. PubMed DOI PMC

Slagle CE, Aoki T, Burdine RD. Nodal-dependent mesendoderm specification requires the combinatorial activities of FoxH1 and Eomesodermin. PLoS Genet. 2011;7:e1002072. doi: 10.1371/journal.pgen.1002072. PubMed DOI PMC

Charney RM, et al. Foxh1 occupies cis -regulatory modules prior to dynamic transcription factor interactions controlling the mesendoderm gene program. Dev. Cell. 2017;40:595–607. doi: 10.1016/j.devcel.2017.02.017. PubMed DOI PMC

Chen X, Rubock MJ, Whitman M. A transcriptional partner for MAD proteins in TGF-β signalling. Nature. 1996;383:691–696. doi: 10.1038/383691a0. PubMed DOI

Germain S, Howell M, Esslemont GM, Hill CS. Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif. Genes Dev. 2000;14:435–451. PubMed PMC

Bruce AEE, et al. The maternally expressed zebrafish T-box gene eomesodermin regulates organizer formation. Development. 2003;130:5503–5517. doi: 10.1242/dev.00763. PubMed DOI

Kunwar PS, et al. Mixer/Bon and FoxH1/Sur have overlapping and divergent roles in Nodal signaling and mesendoderm induction. Development. 2003;130:5589–5599. doi: 10.1242/dev.00803. PubMed DOI

Bjornson CRR, et al. Eomesodermin is a localized maternal determinant required for endoderm induction in Zebrafish. Dev. Cell. 2005;9:523–533. doi: 10.1016/j.devcel.2005.08.010. PubMed DOI

Poulain, M. & Lepage, T. Mezzo, a paired-like homeobox protein is an immediate target of Nodal signalling and regulates endoderm specification in zebrafish. Development129, 4901–4914 (2002). PubMed

Diogo R, et al. A new heart for a new head in vertebrate cardiopharyngeal evolution. Nature. 2015;520:466–473. doi: 10.1038/nature14435. PubMed DOI PMC

Kozmik Z, et al. Characterization of amphioxus Amphivent, an evolutionarily conserved marker for chordate ventral mesoderm. Genesis. 2001;29:172–179. doi: 10.1002/gene.1021. PubMed DOI

Holland ND, Venkatesh TV, Holland LZ, Jacobs DK, Bodmer R. AmphiNk2-tin, an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart. Dev. Biol. 2003;255:128–137. doi: 10.1016/S0012-1606(02)00050-7. PubMed DOI

Holland ND. Formation of the initial kidney and mouth opening in larval amphioxus studied with serial blockface scanning electron microscopy (SBSEM) EvoDevo. 2018;9:16. doi: 10.1186/s13227-018-0104-3. PubMed DOI PMC

Arnold SJ, Hofmann UK, Bikoff EK, Robertson EJ. Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse. Development. 2008;135:501–511. doi: 10.1242/dev.014357. PubMed DOI PMC

Zhang H, Fraser ST, Papazoglu C, Hoatlin ME, Baron MH. Transcriptional activation by the Mixl1 homeodomain protein in differentiating mouse embryonic stem cells. Stem Cells. 2009;27:2884–2895. doi: 10.1634/stemcells.2008-0456. PubMed DOI PMC

Mead PE, Brivanlou IH, Kelley CM, Zon LI. BMP-4-responsive regulation of dorsal–ventral patterning by the homeobox protein Mix.1. Nature. 1996;382:357–360. doi: 10.1038/382357a0. PubMed DOI

Takasato M, et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat. Cell Biol. 2014;16:118–126. doi: 10.1038/ncb2894. PubMed DOI

Costello I, et al. The T-box transcription factor Eomesodermin acts upstream of Mesp1 to specify cardiac mesoderm during mouse gastrulation. Nat. Cell Biol. 2011;13:1084–1091. doi: 10.1038/ncb2304. PubMed DOI PMC

Pfeiffer MJ, et al. Cardiogenic programming of human pluripotent stem cells by dose-controlled activation of EOMES. Nat. Commun. 2018;9:440. doi: 10.1038/s41467-017-02812-6. PubMed DOI PMC

Ormestad M, Astorga J, Carlsson P. Differences in the embryonic expression patterns of mouse Foxf1 and -2 match their distinct mutant phenotypes. Dev. Dyn. 2004;229:328–333. doi: 10.1002/dvdy.10426. PubMed DOI

Martin JF, Olson EN. Identification of a prx1 limb enhancer. Genesis. 2000;26:225–229. doi: 10.1002/(SICI)1526-968X(200004)26:4<225::AID-GENE10>3.0.CO;2-F. PubMed DOI

Reichenbach B, et al. Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish. Dev. Biol. 2008;318:52–64. doi: 10.1016/j.ydbio.2008.02.061. PubMed DOI PMC

De Los Angeles A, Daley GQ. Stem cells: reprogramming in situ. Nature. 2013;502:309–310. doi: 10.1038/nature12559. PubMed DOI

Song K, et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature. 2012;485:599–604. doi: 10.1038/nature11139. PubMed DOI PMC

Lee JH, Protze SI, Laksman Z, Backx PH, Keller GM. Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm populations. Cell Stem Cell. 2017;21:179–194. doi: 10.1016/j.stem.2017.07.003. PubMed DOI

Mendjan S, et al. NANOG and CDX2 pattern distinct subtypes of human mesoderm during exit from pluripotency. Cell Stem Cell. 2014;15:310–325. doi: 10.1016/j.stem.2014.06.006. PubMed DOI

Takeuchi JK, Bruneau BG. Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors. Nature. 2009;459:708–711. doi: 10.1038/nature08039. PubMed DOI PMC

Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132:661–680. doi: 10.1016/j.cell.2008.02.008. PubMed DOI

Zhang G, et al. Comparative genomics reveals insights into avian genome evolution and adaptation. Science. 2014;346:1311–1320. doi: 10.1126/science.1251385. PubMed DOI PMC

Henry GL, Melton DA. Mixer, a homeobox gene required for endoderm development. Science. 1998;281:91–96. doi: 10.1126/science.281.5373.91. PubMed DOI

Kikuchi Y, et al. The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors. Genes Dev. 2000;14:1279–1289. PubMed PMC

Technau U, Scholz CB. Origin and evolution of endoderm and mesoderm. Int. J. Dev. Biol. 2003;47:531–539. PubMed

McEwen GK, et al. Early evolution of conserved regulatory sequences associated with development in vertebrates. PLoS Genet. 2009;5:e1000762. doi: 10.1371/journal.pgen.1000762. PubMed DOI PMC

Parker HJ, Piccinelli P, Sauka-Spengler T, Bronner M, Elgar G. Ancient Pbx-Hox signatures define hundreds of vertebrate developmental enhancers. BMC Genomics. 2011;12:637. doi: 10.1186/1471-2164-12-637. PubMed DOI PMC

Stolfi A, Christiaen L. Genetic and genomic toolbox of the chordate Ciona intestinalis. Genetics. 2012;192:55–66. doi: 10.1534/genetics.112.140590. PubMed DOI PMC

Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Schmid B, et al. High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics. Nat. Commun. 2013;4:2207. doi: 10.1038/ncomms3207. PubMed DOI PMC

Khattak S, et al. Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination. Nat. Protoc. 2014;9:529–540. doi: 10.1038/nprot.2014.040. PubMed DOI

Bordzilovsakya, N. P., Dettlaf, T. A., Duhon, S. T. & Malacinski, G. M. in Developmental Biology of the Axolotl (eds Armstrong, J. B. & Malacinski, G. M.) 201–219 (Oxford University Press, Oxford, 1989).

Parker HJ, Bronner ME, Krumlauf R. A Hox regulatory network of hindbrain segmentation is conserved to the base of vertebrates. Nature. 2014;514:490–493. doi: 10.1038/nature13723. PubMed DOI PMC

Kuraku S, Takio Y, Sugahara F, Takechi M, Kuratani S. Evolution of oropharyngeal patterning mechanisms involving Dlx and endothelins in vertebrates. Dev. Biol. 2010;341:315–323. doi: 10.1016/j.ydbio.2010.02.013. PubMed DOI

Davidson B, Shi W, Levine M. Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis. Development. 2005;132:4811–4818. doi: 10.1242/dev.02051. PubMed DOI

Racioppi C, et al. Fibroblast growth factor signalling controls nervous system patterning and pigment cell formation in Ciona intestinalis. Nat. Commun. 2014;5:4830. doi: 10.1038/ncomms5830. PubMed DOI PMC

Kozmikova I, Kozmik Z. Gene regulation in amphioxus: An insight from transgenic studies in amphioxus and vertebrates. Mar. Genomics. 2015;24:159–166. doi: 10.1016/j.margen.2015.06.003. PubMed DOI

Fuentes M, et al. Insights into spawning behavior and development of the european amphioxus (Branchiostoma lanceolatum) J. Exp. Zool. B Mol. Dev. Evol. 2007;308B:484–493. doi: 10.1002/jez.b.21179. PubMed DOI

Cell type and regulatory analysis in amphioxus illuminates evolutionary origin of the vertebrate head

Conserved enhancers control notochord expression of vertebrate Brachyury

Conserved enhancer logic controls the notochord expression of vertebrate Brachyury