title: The Evx1/Evx1as gene locus regulates anterior-posterior patterning during gastrulation.
abstract: Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis.
authors.affiliation: Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, 4101, Australia.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Bell
authors.affiliation: The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Paulo P
authors.initials: PP
authors.lastname: Amaral
authors.affiliation: The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.|Garvan Institute of Medical Research, Sydney, Australia.
authors.forename: Anton
authors.initials: A
authors.lastname: Kalsbeek
authors.affiliation: Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, 4101, Australia.
authors.forename: Graham W
authors.initials: GW
authors.lastname: Magor
authors.affiliation: Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, 4101, Australia.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Gillinder
authors.affiliation: The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Pierre
authors.initials: P
authors.lastname: Tangermann
authors.affiliation: Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, 4101, Australia.
authors.forename: Lorena
authors.initials: L
authors.lastname: di Lisio
authors.affiliation: Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, 4101, Australia.|Diamantina Institute; Translational Research Institute, University of Queensland, Brisbane, Queensland, 4102, Australia.
authors.forename: Seth W
authors.initials: SW
authors.lastname: Cheetham
authors.affiliation: Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, 4101, Australia.|Diamantina Institute; Translational Research Institute, University of Queensland, Brisbane, Queensland, 4102, Australia.
authors.forename: Franziska
authors.initials: F
authors.lastname: Gruhl
authors.affiliation: The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.|The Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, 4102, Australia.
authors.forename: Jessica
authors.initials: J
authors.lastname: Frith
authors.affiliation: Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, 4101, Australia.|The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Tallack
authors.affiliation: The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.|The Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, 4102, Australia.
authors.forename: Ke-Lin
authors.initials: KL
authors.lastname: Ru
authors.affiliation: The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Joanna
authors.initials: J
authors.lastname: Crawford
authors.affiliation: Garvan Institute of Medical Research, Sydney, Australia.|St Vincents Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: John S
authors.initials: JS
authors.lastname: Mattick
authors.affiliation: Garvan Institute of Medical Research, Sydney, Australia.|Diamantina Institute; Translational Research Institute, University of Queensland, Brisbane, Queensland, 4102, Australia.|St Vincents Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia.
authors.forename: Marcel E
authors.initials: ME
authors.lastname: Dinger
authors.affiliation: Mater Research, Translational Research Institute, University of Queensland, Brisbane, Queensland, 4101, Australia.|The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.|The Princess Alexandra Hospital, Brisbane, Queensland, 4102, Australia.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Perkins
mesh_terms: D000818:Animals; D019521:Body Patterning / Q000502:physiology*; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology*; D004622:Embryo, Mammalian / Q000196:embryology*; D054262:Gastrulation / Q000502:physiology*; D000072669:Gene Editing; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D062085:RNA, Long Noncoding / Q000096:biosynthesis* / Q000235:genetics; D060509:Wnt3A Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; C489555:Evx1 protein, mouse; D018398:Homeodomain Proteins; D062085:RNA, Long Noncoding; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse