NFIX - one gene, two knockouts, multiple effects

Minireview NFIX - one gene, two knockouts, multiple effects Vladimir Pekarik* and Juan Carlos Izpisua Belmonte*† Address: *Center of Regenerative Medicine in Barcelona, Doctor Aiguader 88, 08003 Barcelona, Spain. †Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. Correspondence: Juan Carlos Izpisua Belmonte. Email: ; Published: 23 October 2008 Journal of Biology 2008, 7:29 (doi:10.1186/jbiol94) The electronic version of this article is the complete one and can be found online at http://jbiol.com/content/7/8/29 © 2008 BioMed Central Ltd Abstract A previous knockout of the transcription factor gene nuclear factor IX (NFIX) in mice produced impaired development of the corpus callosum and severe skeletal defects. A recent paper in BMC Developmental Biology reports an apparently similar NFIX knockout that produced marked differences in phenotype, raising intriguing general questions about the possible causes of such differences in mouse knockouts. The Nuclear Factor I (NFI) family of evolutionarily conserved transcription factors is widely expressed during development and in adulthood, in mammals but has mainly been studied in respect to brain development, where it is intimately associated with glial function [1,2]. The family consists of four members, NFIA, NFIB, NFIC and NFIX, each having multiple splice variants [3]. NFI proteins can directly bind to the promoter and regulate the transcription activity of glial fibrillar acidic protein (GFAP), a marker of glial cells [4]. Different members of the family have been shown to have a variety of roles in neural development but taken together, loss-of-function studies of NFI members in mice reveal a common theme - a lack of development (agenesis) of the corpus callosum, the large tract of nerve fibers interconnecting the left and right hemispheres. The main feature of corpus callosum agenesis is an inability to perform tasks where a matching of visual patterns is required, for example face processing, which in turn results in social difficulties. In mild cases intelligence is mainly unaffected but low muscle tone and motor coordination are affected. In severe cases intellectual retardation, hydrocephalus, seizures and spasticity might be involved. The effect of a mutation varies from partial callosal agenesis (in the case of loss of function of NFIX) to severe agenesis (with loss of function of NFIB having a greater effect than loss of NFIA, as described later). Less is known so far about the actions of the NFIX gene than about the other members of the family. One known property of NFIX is the regulation of expression of astrocytespecific α1-antichymotrypsin [5]. To determine the effects of loss of function of NFIX, two groups have recently described knockouts of the NFIX gene [6,7]. Their results turned out to be surprisingly different. The first knockout was reported by a team at the University of Freiburg (Driller et al. [6]) while the second was generated by a group from the University of New York at Buffalo and described in BMC Developmental Biology (Campbell et al. [7]). Here, we briefly review some of the possible reasons for such discrepancies. For simplicity, we will call the mutant strain generated in Freiburg ‘X-Freiburg’ and the one generated in New York ‘X-NY’. Animals of the X-Freiburg strain suffered from hydrocephalus, partial agenesis of the corpus callosum, and spinal deformities that were due to a delay in ossification of vertebral bodies and progressive degeneration of intervertebral discs. Femoral defects were also noticed and animals usually died at around postnatal day Journal of Biology 2008, 7:29 29.2 Journal of Biology 2008, Volume 7, Article 29 (a) NFI wild-type allele Pekarik and Izpisua Belmonte 3′ splice acceptor 5′ splice acceptor 2 1a/1b Transcript (b) Gene knockouts X-Freiburg http://jbiol.com/content/7/8/29 1 3 3 2 3′ splice acceptor 1a/1b 3 LacZ 3 1 + 1 2 loxP X-NY 1a/1b 3 1 3 5′ splice acceptor NFIA 1a/1b 3 PGK-neo 1 3 + 3′ splice acceptor NFIB-NY LacZ 3 LacZ-neo 3 1a/1b 1 3 + 1 3′ splice acceptor NFIB-Freiburg NFIC 1a/1b 1 3 1 3 1a/1b + 1 PGK-neo 3 + Figure 1 For simplicity the same structure is drawn for all four NFI genes. (a) The organization of the NFI genes. They can all use an alternative exon 1, here denoted as a single box labeled 1a/1b. The DNA-binding and dimerization domains are located in exon 2. (b) In general, two approaches are used for knockouts of these genes. The first relies on complete deletion of the second exon (including 5’ and 3’ splice acceptor sites of proximal introns), as shown here in the X-NY knockout. The second strategy is to insert LacZ (or a LacZ-neo hybrid or PGK-neo hybrid) in-frame into the second exon, leading to production of a fusion protein composed of a few amino acids derived from exons 1 and 2 of the NFI and LacZ genes. In all cases an alternative splice variant joining the first and the third exon of the NFI gene will be formed. The third exon is not in frame with the first, and so premature termination of translation will occur. Whether a peptide produced from the joining of exons 1 and 3 has any physiological function was never analyzed, but judging from the very different phenotypes of the different knockout strains it seems rather unlikely. The NFIB constructs are reported in [15,16], the NFIA knockout in [17] and the NFIC knockout in [18]. (P) 21-28. The X-NY strain, on the other hand, did not suffer from such severe impairments. Callosal agenesis as seen in the X-Freiburg strain was not noted in X-NY NFIX-/animals. The cingulate cortex and the entire brain are expanded along the dorsal-ventral axis, hippocampus formation is aberrant, and overabundant Pax6- and doublecortinpositive cells are found in the lateral ventricles of X-NY mice. When the X-NY mice were fed with a soft dough chow they showed a lag in weight gain compared to non-mutant animals, but after P20 the growth rate increased and a few of the animals survived to adulthood. Skeletal deformities observed by Driller et al. and absent in the animals reported by Campbell et al. can be attributed to the severe malnutrition, which was relieved by Campbell et al. by the change in diet. Another possibility is that brain development abnormalities result in reduced appetite, leading again to skeletal defects. Reconciling the differences How can the discrepancies reported between the two NFIX-/strains be reconciled? Among various possible explanations, one could be an alteration of neighboring gene expression. A case in point is the sequential generation of several prion protein (PrP) knockout strains that showed profoundly different phenotypes. Only later was this variation proved to be due to the unintentional activation of another gene in the vicinity of the PrP gene, later named Doppel [8], and which was shown to be neurotoxic. Both reports of the NFIX knockouts [6,7] describe t (...truncated)