Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development.
BACKGROUND
Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development.


RESULTS
By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage.


CONCLUSIONS
We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Leo
L
Kurian
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Aitor
A
Aguirre
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Ignacio
I
Sancho-Martinez
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Christopher
C
Benner
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Tomoaki
T
Hishida
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Thai B
TB
Nguyen
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Pradeep
P
Reddy
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Emmanuel
E
Nivet
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Marie N
MN
Krause
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
David A
DA
Nelles
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Concepcion Rodriguez
CR
Esteban
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Josep M
JM
Campistol
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Gene W
GW
Yeo
Gene Expression Laboratory (L.K., A.A., I.S.-M., T.H., T.B.N., P.R., E.N., M.N.K., C.R.E., J.C.I.B.) and Integrative Genomics Core (C.B.), Salk Institute for Biological Studies, La Jolla, CA; University of California San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, La Jolla (L.K., T.B.N., D.A.N., G.W.Y.); and Hospital Clinic, University of Barcelona, IDIBAPS, Barcelona, Spain (J.M.C.).
Juan Carlos Izpisua
JCI
Belmonte
D000818:Animals; D002319:Cardiovascular System / Q000254:growth & development* / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D002874:Chromosome Mapping; D047108:Embryonic Development / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D042783:Endothelial Cells / Q000737:chemistry*; D005318:Fetal Heart / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D060172:Morpholinos / Q000493:pharmacokinetics; D032383:Myocytes, Cardiac / Q000737:chemistry* / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000737:chemistry*; D062085:RNA, Long Noncoding / Q000302:isolation & purification* / Q000502:physiology; D017423:Sequence Analysis, RNA; D059467:Transcriptome; D014714:Vertebrates / Q000235:genetics* / Q000254:growth & development; D015027:Zebrafish / Q000196:embryology
D060172:Morpholinos; D062085:RNA, Long Noncoding
RNA, long noncoding; cardiovascular system; growth and development; transcriptome; vertebrates