title: An Lmx1b-miR135a2 regulatory circuit modulates Wnt1/Wnt signaling and determines the size of the midbrain dopaminergic progenitor pool.
abstract: MicroRNAs regulate gene expression in diverse physiological scenarios. Their role in the control of morphogen related signaling pathways has been less studied, particularly in the context of embryonic Central Nervous System (CNS) development. Here, we uncover a role for microRNAs in limiting the spatiotemporal range of morphogen expression and function. Wnt1 is a key morphogen in the embryonic midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, which modulates the extent of Wnt1/Wnt signaling and the size of the dopamine progenitor domain. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain size and dopamine progenitor allocation. Conditional removal of Lmx1b has the opposite effect, in that expansion of the dopamine progenitor domain is severely compromised. Next, we provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in embryonic stem cells (ESCs) results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of miR135a2 during an early window in vivo phenocopies the Lmx1b conditional knockout. When En1::Cre, but not Shh::Cre or Nes::Cre, is used for recombination, the expansion of Lmx1a/b+ progenitors is selectively reduced. Bioinformatics and luciferase assay data suggests that miR135a2 targets Lmx1b and many genes in the Wnt signaling pathway, including Ccnd1, Gsk3b, and Tcf7l2. Consistent with this, we demonstrate that this mutant displays reductions in the size of the Lmx1b/Wnt1 domain and range of canonical Wnt signaling. We posit that microRNA modulation of the Lmx1b/Wnt axis in the early midbrain/isthmus could determine midbrain size and allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for programming ESCs towards a dopaminergic fate in vitro, these studies could impact the rational design of such protocols.
authors.affiliation: Northwestern University Feinberg School of Medicine, Department of Neurology and Center for Genetic Medicine, Chicago, Illinois, United States of America.
authors.forename: Angela
authors.initials: A
authors.lastname: Anderegg
authors.affiliation: Northwestern University Feinberg School of Medicine, Department of Neurology and Center for Genetic Medicine, Chicago, Illinois, United States of America.
authors.forename: Hsin-Pin
authors.initials: HP
authors.lastname: Lin
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Jun-An
authors.initials: JA
authors.lastname: Chen
authors.affiliation: Northwestern University Feinberg School of Medicine, Department of Neurology and Center for Genetic Medicine, Chicago, Illinois, United States of America.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Caronia-Brown
authors.affiliation: Northwestern University Feinberg School of Medicine, Department of Neurology and Center for Genetic Medicine, Chicago, Illinois, United States of America.
authors.forename: Natalya
authors.initials: N
authors.lastname: Cherepanova
authors.affiliation: Northwestern University Feinberg School of Medicine, Department of Neurology and Center for Genetic Medicine, Chicago, Illinois, United States of America.
authors.forename: Beth
authors.initials: B
authors.lastname: Yun
authors.affiliation: Northwestern University Feinberg School of Medicine, Department of Neurology and Center for Genetic Medicine, Chicago, Illinois, United States of America.
authors.forename: Milan
authors.initials: M
authors.lastname: Joksimovic
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Jason
authors.initials: J
authors.lastname: Rock
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Harfe
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, United States of America.
authors.forename: Randy
authors.initials: R
authors.lastname: Johnson
authors.affiliation: Northwestern University Feinberg School of Medicine, Department of Neurology and Center for Genetic Medicine, Chicago, Illinois, United States of America.
authors.forename: Rajeshwar
authors.initials: R
authors.lastname: Awatramani
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000166:cytology / Q000378:metabolism; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D060850:LIM-Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D008636:Mesencephalon / Q000254:growth & development / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D055495:Neurogenesis / Q000235:genetics*; D010300:Parkinson Disease / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D043244:Ribonuclease III / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000235:genetics; D051155:Wnt1 Protein / Q000235:genetics*
chemical_list: C112149:LIM homeobox transcription factor 1 beta; D060850:LIM-Homeodomain Proteins; D035683:MicroRNAs; D014157:Transcription Factors; D051155:Wnt1 Protein; C495246:Wnt1 protein, mouse; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases; D004298:Dopamine