title: A trans-Regulatory Code for the Forebrain Expression of Six3.2 in the Medaka Fish.
abstract: A well integrated and hierarchically organized gene regulatory network is responsible for the progressive specification of the forebrain. The transcription factor Six3 is one of the central components of this network. As such, Six3 regulates several components of the network, but its upstream regulators are still poorly characterized. Here we have systematically identified such regulators, taking advantage of the detailed functional characterization of the regulatory region of the medaka fish Six3.2 ortholog and of a time/cost-effective trans-regulatory screening, which complemented and overcame the limitations of in silico prediction approaches. The candidates resulting from this search were validated with dose-response luciferase assays and expression pattern criteria. Reconfirmed candidates with a matching expression pattern were also tested with chromatin immunoprecipitation and functional studies. Our results confirm the previously proposed direct regulation of Pax6 and further demonstrate that Msx2 and Pbx1 are bona fide direct regulators of early Six3.2 distribution in distinct domains of the medaka fish forebrain. They also point to other transcription factors, including Tcf3, as additional regulators of different spatial-temporal domains of Six3.2 expression. The activity of these regulators is discussed in the context of the gene regulatory network proposed for the specification of the forebrain.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, c/ Nicolas Cabrera 1, Madrid 28049, Spain,; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), c/ Nicolas Cabrera 1, Madrid 28049, Spain; Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avda. Dr. Arce 37, Madrid, 28002, Spain,. Electronic address: leonardo.beccari@unige.ch.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Beccari
authors.affiliation: Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, c/ Nicolas Cabrera 1, Madrid 28049, Spain,; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), c/ Nicolas Cabrera 1, Madrid 28049, Spain; Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avda. Dr. Arce 37, Madrid, 28002, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Marco-Ferreres
authors.affiliation: Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, c/ Nicolas Cabrera 1, Madrid 28049, Spain,; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), c/ Nicolas Cabrera 1, Madrid 28049, Spain; Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avda. Dr. Arce 37, Madrid, 28002, Spain.
authors.forename: Noemi
authors.initials: N
authors.lastname: Tabanera
authors.affiliation: Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avda. Dr. Arce 37, Madrid, 28002, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Manfredi
authors.affiliation: the Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Souren
authors.affiliation: the Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Wittbrodt
authors.affiliation: Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avda. Dr. Arce 37, Madrid, 28002, Spain,; the Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, Pozzuoli, Naples, 80078, Italy.
authors.forename: Ivan
authors.initials: I
authors.lastname: Conte
authors.affiliation: the Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Wittbrodt
authors.affiliation: Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, c/ Nicolas Cabrera 1, Madrid 28049, Spain,; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), c/ Nicolas Cabrera 1, Madrid 28049, Spain; Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avda. Dr. Arce 37, Madrid, 28002, Spain,. Electronic address: pbovolenta@cbm.csic.es.
authors.forename: Paola
authors.initials: P
authors.lastname: Bovolenta
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019521:Body Patterning / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics*; D029941:Fish Proteins / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D053263:Gene Regulatory Networks; D018398:Homeodomain Proteins / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009990:Oryzias / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D016548:Prosencephalon / Q000196:embryology* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D000097607:Homeobox Protein SIX3
chemical_list: D005136:Eye Proteins; D029941:Fish Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D000097607:Homeobox Protein SIX3; C084119:MSX2 protein
keywords: HCNC; brain; gene regulation; neurodevelopment; transcription factor; transcription regulation; transcriptional enhancers