The establishment of neuronal properties is controlled by Sox4 and Sox11.

The progression of neurogenesis relies on proneural basic helix-loop-helix (bHLH) transcription factors. These factors operate in undifferentiated neural stem cells and induce cell cycle exit and the initiation of a neurogenic program. However, the transient expression of proneural bHLH proteins in neural progenitors indicates that expression of neuronal traits must rely on previously unexplored mechanisms operating downstream from proneural bHLH proteins. Here we show that the HMG-box transcription factors Sox4 and Sox11 are of critical importance, downstream from proneural bHLH proteins, for the establishment of pan-neuronal protein expression. Examination of a neuronal gene promoter reveals that Sox4 and Sox11 exert their functions as transcriptional activators. Interestingly, the capacity of Sox4 and Sox11 to induce the expression of neuronal traits is independent of mechanisms regulating the exit of neural progenitors from the cell cycle. The transcriptional repressor protein REST/NRSF has been demonstrated to block neuronal gene expression in undifferentiated neural cells. We now show that REST/NRSF restricts the expression of Sox4 and Sox11, explaining how REST/NRSF can prevent precocious expression of neuronal proteins. Together, these findings demonstrate a central regulatory role of Sox4 and Sox11 during neuronal maturation and mechanistically separate cell cycle withdrawal from the establishment of neuronal properties.

[1]  François Guillemot,et al.  Proneural genes and the specification of neural cell types , 2002, Nature Reviews Neuroscience.

[2]  R. Ebright,et al.  Mechanisms of viral activators. , 1998, Cold Spring Harbor symposia on quantitative biology.

[3]  K. Tomita,et al.  Mammalian achaete–scute and atonal homologs regulate neuronal versus glial fate determination in the central nervous system , 2000, The EMBO journal.

[4]  M. Tsai,et al.  Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice. , 1997, Genes & development.

[5]  Xiaohong Liu,et al.  Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors , 2003, Nature.

[6]  J. B. Jaynes,et al.  A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and msh-class homeoproteins, mediates active transcriptional repression in vivo. , 1996, Development.

[7]  T. Jessell,et al.  A Homeodomain Protein Code Specifies Progenitor Cell Identity and Neuronal Fate in the Ventral Neural Tube , 2000, Cell.

[8]  B. Novitch,et al.  Vertebrate neurogenesis is counteracted by Sox1–3 activity , 2003, Nature Neuroscience.

[9]  Ernst R. Tamm,et al.  Gene Targeting Reveals a Widespread Role for the High-Mobility-Group Transcription Factor Sox11 in Tissue Remodeling , 2004, Molecular and Cellular Biology.

[10]  F. Rajaii,et al.  In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo , 2004, Developmental dynamics : an official publication of the American Association of Anatomists.

[11]  L. Pevny,et al.  SOX2 Functions to Maintain Neural Progenitor Identity , 2003, Neuron.

[12]  M. Greenberg,et al.  Neurogenin Promotes Neurogenesis and Inhibits Glial Differentiation by Independent Mechanisms , 2001, Cell.

[13]  S. Moody,et al.  The development of acetylcholinesterase activity in the embryonic nervous system of the frog, Xenopus laevis. , 1988, Brain research.

[14]  F. Guillemot,et al.  A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons. , 2000, Genes & development.

[15]  T. Perlmann,et al.  Defining Requirements for Heterodimerization between the Retinoid X Receptor and the Orphan Nuclear Receptor Nurr1* , 2002, The Journal of Biological Chemistry.

[16]  J. Olson,et al.  Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. , 2000, Development.

[17]  M. Ballivet,et al.  NeuroM, a neural helix-loop-helix transcription factor, defines a new transition stage in neurogenesis. , 1997, Development.

[18]  R. Lovell-Badge,et al.  A role for SOX 1 in neural determination , 1998 .

[19]  Magnus Sandberg,et al.  Sox21 promotes the progression of vertebrate neurogenesis , 2005, Nature Neuroscience.

[20]  A. Rizzino,et al.  Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity* , 2003, The Journal of Biological Chemistry.

[21]  P. Ward,et al.  Conservation of Sox4 gene structure and expression during chicken embryogenesis. , 2003, Gene.

[22]  F. Guillemot,et al.  Restricted expression of a novel murine atonal-related bHLH protein in undifferentiated neural precursors. , 1996, Developmental biology.

[23]  T. Jessell,et al.  Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes , 1994, Cell.

[24]  H. Niwa,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector. , 1991, Gene.

[25]  David J. Anderson,et al.  Comparison of the generic neuronal differentiation and neuron subtype specification functions of mammalian achaete-scute and atonal homologs in cultured neural progenitor cells. , 2002, Development.

[26]  D J Anderson,et al.  The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes , 1995, Science.

[27]  S. Moody,et al.  Cloning and characterization of the 5'-flanking region of the rat neuron-specific Class III beta-tubulin gene. , 2002, Gene.

[28]  J. Arthur,et al.  Nur77 is phosphorylated in cells by RSK in response to mitogenic stimulation. , 2006, The Biochemical journal.

[29]  Sing-Hoi Sze,et al.  Corepressor-Dependent Silencing of Chromosomal Regions Encoding Neuronal Genes , 2002, Science.

[30]  H. Kondoh,et al.  Pairing SOX off: with partners in the regulation of embryonic development. , 2000, Trends in genetics : TIG.

[31]  J. Postlethwait,et al.  Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development , 2000, Developmental dynamics : an official publication of the American Association of Anatomists.

[32]  H. Clevers,et al.  Sox‐4, an Sry‐like HMG box protein, is a transcriptional activator in lymphocytes. , 1993, The EMBO journal.

[33]  M. Wegner,et al.  From head to toes: the multiple facets of Sox proteins. , 1999, Nucleic acids research.

[34]  M. Wegner,et al.  Cooperative Function of POU Proteins and SOX Proteins in Glial Cells* , 1998, The Journal of Biological Chemistry.

[35]  H Clevers,et al.  Sequence-specific high mobility group box factors recognize 10-12-base pair minor groove motifs. , 2000, The Journal of biological chemistry.

[36]  P. Koopman,et al.  Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio‐mesenchymal induction , 1997, Developmental dynamics : an official publication of the American Association of Anatomists.

[37]  R. Grosschedl,et al.  Ebf1 controls early cell differentiation in the embryonic striatum. , 1999, Development.

[38]  C. Kintner,et al.  Neurogenesis in Embryos and in Adult Neural Stem Cells , 2002, The Journal of Neuroscience.

[39]  H. Weintraub,et al.  Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. , 1995, Science.

[40]  E. Cartwright,et al.  Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development , 1995, Mechanisms of Development.

[41]  Gail Mandel,et al.  REST: A mammalian silencer protein that restricts sodium channel gene expression to neurons , 1995, Cell.

[42]  F. Guillemot,et al.  The bHLH Protein NEUROGENIN 2 Is a Determination Factor for Epibranchial Placode–Derived Sensory Neurons , 1998, Neuron.

[43]  W. Harris,et al.  X-ngnr-1 and Xath3 promote ectopic expression of sensory neuron markers in the neurula ectoderm and have distinct inducing properties in the retina. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[44]  M. Wegner,et al.  Sox8 Is a Specific Marker for Muscle Satellite Cells and Inhibits Myogenesis* , 2003, Journal of Biological Chemistry.

[45]  K. Haglid,et al.  A Rapid HPLC Method to Separate the Triplet Proteins of Neurofilament , 1987, Journal of neurochemistry.

[46]  C. Mello,et al.  RNAi (Nematodes: Caenorhabditis elegans). , 2002, Advances in genetics.

[47]  A. Paquette,et al.  NRSF/REST is required in vivo for repression of multiple neuronal target genes during embryogenesis , 1998, Nature Genetics.

[48]  H Clevers,et al.  Roles of Sox4 in central nervous system development. , 2000, Brain research. Molecular brain research.

[49]  R. Lovell-Badge,et al.  A role for SOX1 in neural determination. , 1998, Development.

[50]  T. Jessell,et al.  Coordinate Regulation of Motor Neuron Subtype Identity and Pan-Neuronal Properties by the bHLH Repressor Olig2 , 2001, Neuron.

[51]  J. Nardelli,et al.  The GATA2 transcription factor negatively regulates the proliferation of neuronal progenitors , 2006, Development.

[52]  L. Pevny,et al.  SOX genes and neural progenitor identity , 2005, Current Opinion in Neurobiology.

[53]  J. Martial,et al.  sox4b is a key player of pancreatic alpha cell differentiation in zebrafish. , 2005, Developmental biology.

[54]  A. Matus,et al.  Differential expression of distinct microtubule-associated proteins during brain development. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[55]  S. Garel,et al.  Ebf gene function is required for coupling neuronal differentiation and cell cycle exit , 2003, Development.

[56]  A. Joyner,et al.  Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system , 1993, Mechanisms of Development.

[57]  Y. Yokota.  Id and development , 2001, Oncogene.

[58]  David J. Anderson,et al.  SOX10 Maintains Multipotency and Inhibits Neuronal Differentiation of Neural Crest Stem Cells , 2003, Neuron.