Paraformaldehyde fixed frozen section of adult rat brain stem stained with RPCA-Fox3 in red and counterstained for MAP2 with MCA-5H11 in green. DNA is shown in blue. Note that RPCA-Fox3 stains neuronal nuclei and distal perikarya and that the MAP2 antibody stains the dendrites extending from these cells. RPCA-Fox3 stains exactly like the original NeuN monoclonal antibody and like EnCor NeuN/Fox3 mouse monoclonal, MCA-1B7. These antibodies do not bind to the nuclei of perikarya of any non-neuronal cells, so that they can be used to identify and quantify neurons.
Fox3 is one of a family of 3 mammalian Fox homologues. Fox was discovered in C. elegans as a gene involved in sex determination, and the name Fox is an acronym of “Feminizing locus on X”.
The Fox protein and its 3 mammalian homologues are all about 46 kDa proteins each of which includes a central highly conserved RRM type RNA recognition motif, which corresponds to a small ~70 amino acid structure consisting of 4 beta strands and two alpha-helices. An alternate name for Fox 3 is hexaribonucleotide binding protein 3, and these proteins are believed to have a role in the regulation of mRNA splicing.
Much interest has focused on Fox3 as a result of the recent finding that this protein corresponds to NeuN, a neuronal nuclear antigen. NeuN was first described in 1994 by Mullen et al. (2), who raised a series of monoclonal antibodies to mouse antigens with the original intent of finding mouse species specific markers which would be useful in transplantation experiments. In the event they obtained a hybridoma clone, called mAb A60, which proved to bind an antigen expressed only in neuronal nuclei, and which appeared to work on all vertebrates. A few neuronal cell types were not recognized by the NeuN antibody, such as Purkinje cells in the cerebellum and photoreceptors in the retina, but the vast majority of neurons were, making this antibody very useful for the identification of neurons. The protein bound by this antibody was not characterized, though the molecular weight of this protein was shown to be three closely spaced bands running at 46-48 kDa on SDS-PAGE gels. The exact identity of the NeuN protein was not elucidated, despite several attempts, for many years later. Despite this the mAb A66 antibody has become very widely used as a robust marker of neurons and neuronal stem cells.
Recently Kim et al. showed that NeuN corresponds to Fox3 (3). Fox3 is therefore a protein which has a function in RNA splicing and is expressed heavily and specifically in neuronal nuclei.
Our antibody was raised against the N-terminal 100 amino acids of human Fox3 as expressed in and purified from E. coli. We did not use full length Fox3 as immunogen since the three mammalian Fox1 homologues, namely Fox1, Fox2 and Fox3, include virtually identical RRM motifs. The N-terminal region of the three molecules are much more variable in the three molecules so antibodies specific for each of the three molecules can therefore be generated. The HGNC name for this protein is RBFOX3.
1. Hodgkin J, Zellan JD, Albertson DG. Identification of a candidate primary sex determination locus, fox-1, on the X chromosome of Caenorhabditis elegans. Development 120:3681-3689 (1994).
2. Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201-211 (1994).
3. Kim KK, Adelstein RS, Kawamoto S. Identification of neuronal nuclei (NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors. J. Biol. Chem. 284:31052-31061 (2009).
4. Underwood,J.G., Boutz,P.L., Dougherty,J.D., Stoilov,P. and Black,D.L. Homologues of the Caenorhabditis elegans Fox-1 protein are neuronal splicing regulators in mammals. Mol. Cell. Biol. 25:10005-10016 (2005).