title: Expression and functional analysis of the Wnt/beta-catenin induced mir-135a-2 locus in embryonic forebrain development.
abstract: BACKGROUND
  Brain size and patterning are dependent on dosage-sensitive morphogen signaling pathways - yet how these pathways are calibrated remains enigmatic. Recent studies point to a new role for microRNAs in tempering the spatio-temporal range of morphogen functions during development. Here, we investigated the role of miR-135a, derived from the mir-135a-2 locus, in embryonic forebrain development.
  
  
  METHODS
  1. We characterized the expression of miR-135a, and its host gene Rmst, by in situ hybridization (ish). 2. We conditionally ablated, or activated, beta-catenin in the dorsal forebrain to determine if this pathway was necessary and/or sufficient for Rmst/miR-135a expression. 3. We performed bioinformatics analysis to unveil the most predicted pathways targeted by miR-135a. 4. We performed gain and loss of function experiments on mir-135a-2 and analyzed by ish the expression of key markers of cortical hem, choroid plexus, neocortex and hippocampus.
  
  
  RESULTS
  1. miR-135a, embedded in the host long non-coding transcript Rmst, is robustly expressed, and functional, in the medial wall of the embryonic dorsal forebrain, a Wnt and TGFβ/BMP-rich domain. 2. Canonical Wnt/beta-catenin signaling is critical for the expression of Rmst and miR-135a, and the cortical hem determinant Lmx1a. 3. Bioinformatics analyses reveal that the Wnt and TGFβ/BMP cascades are among the top predicted pathways targeted by miR-135a. 4. Analysis of mir-135a-2 null embryos showed that dorsal forebrain development appeared normal. In contrast, modest mir-135a-2 overexpression, in the early dorsal forebrain, resulted in a phenotype resembling that of mutants with Wnt and TGFβ/BMP deficits - a smaller cortical hem and hippocampus primordium associated with a shorter neocortex as well as a less convoluted choroid plexus. Interestingly, late overexpression of mir-135a-2 revealed no change.
  
  
  CONCLUSIONS
  All together, our data suggests the existence of a Wnt/miR-135a auto-regulatory loop, which could serve to limit the extent, the duration and/or intensity of the Wnt and, possibly, the TGFβ/BMP pathways.
authors.affiliation: Department of Neurology and Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, 7-113 Lurie Bldg., 303 E. Superior Street, Chicago, IL, 60611, USA. giuliana.caronia-brown@northwestern.edu.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Caronia-Brown
authors.affiliation: Department of Neurology and Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, 7-113 Lurie Bldg., 303 E. Superior Street, Chicago, IL, 60611, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Anderegg
authors.affiliation: Department of Neurology and Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, 7-113 Lurie Bldg., 303 E. Superior Street, Chicago, IL, 60611, USA.
authors.forename: Rajeshwar
authors.initials: R
authors.lastname: Awatramani
mesh_terms: D000818:Animals; D060850:LIM-Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D016548:Prosencephalon / Q000196:embryology* / Q000378:metabolism*; D055411:TGF-beta Superfamily Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D060449:Wnt Signaling Pathway
chemical_list: D060850:LIM-Homeodomain Proteins; C489692:Lmx1a protein, mouse; D035683:MicroRNAs; C549289:Mirn135 microRNA, mouse; D055411:TGF-beta Superfamily Proteins; D014157:Transcription Factors
keywords: Forebrain; Lmx1a; Rmst; Wnts; beta-catenin; miR-135a