Nuclear Factor One X Regulates Bobby Sox During Development of the Mouse Forebrain

Abstract

The transcription factor nuclear factor one X (NFIX) plays a central role during the development of the neocortex and hippocampus, through the activation of astrocyte-specific gene expression and the repression of progenitor-specific pathways. However, our understanding of transcriptional targets of NFIX during cortical development remains limited. Here, we identify the transcription factor Bobby sox (Bbx) as a target for NFI-mediated transcriptional control. BBX is expressed within ventricular zone progenitor cells within the developing neocortex and hippocampus, and its expression is upregulated in Nfix ^−/− mice. Moreover, we reveal that NFIX can repress Bbx promoter-driven expression. Collectively, these data suggest that Bbx is a downstream target of NFIX during development of the forebrain.

Introduction

The transcription factors of the NFI family (NFIA, NFIB, NFIC and NFIX) are emerging as important regulators of neural development. NFIX is expressed widely within the developing brain, including within the ventricular zone and dentate gyrus of the embryonic hippocampus, and Nfix ^−/− mice exhibit abnormal development of the neocortex, hippocampus and cerebellum (Campbell et al. 2008; Piper et al. 2011). At a mechanistic level, NFIX has been shown to promote the expression of astrocyte-specific genes in human glioblastoma-derived cell lines (Brun et al. 2009) and to repress expression of the progenitor cell-specific gene Sox9 in vivo (Heng et al. 2013). As such, mice lacking Nfix exhibit prolonged progenitor cell self-renewal, concomitant with delayed neuronal and glial differentiation within the hippocampus and neocortex (Heng et al. 2013).

Despite these findings, our understanding of the genes regulated by NFIX during development of the forebrain remains limited. To identify further potential targets of NFIX, we used a multifaceted approach. Firstly, we mined a recently published microarray performed on the hippocampus of embryonic day (E) 16 Nfix ^−/− mice to identify transcription factors that were misregulated in the mutant mice (Heng et al. 2013). We then ascertained if potential targets were expressed within the developing forebrain using gene expression data within the Allen Brain Atlas. Finally, we used an in silico bioinformatic approach to identify whether these putative NFIX target genes contained an NFI-binding motif close to their transcription start site. This analysis identified Bobby sox (also called Hbp2) as a potential target downstream of NFIX. Here, we reveal that BBX is expressed by neural progenitor cells within the developing neocortex and hippocampus, and that there are significantly more cells expressing BBX in the neocortical and hippocampal ventricular zone of E16 Nfix ^−/− mice. Finally, we demonstrate that NFIX can regulate Bbx promoter-driven transcriptional activity, suggestive of NFIX-regulating Bbx transcription during forebrain development.

Methods

Mouse Strains

C57Bl/6 J Nfix ^+/+ and Nfix ^−/− littermate mice were used in this study. The animals were bred at the University of Queensland under approval from the institutional Animal Ethics Committee.

Immunohistochemistry

Immunohistochemistry using the chromogen 3,3′ diaminobenzidine on floating vibratome sections (50 μm) was performed as described previously (Heng et al. 2013) using a rabbit-anti-BBX antibody (Abcam, ab94418, 1/10,000). Immunohistochemistry was performed on 6 μm paraffin sections as described previously (Barry et al. 2008) using the anti-BBX antibody (1/1,000). For all immunohistochemical analyses, at least 5 littermate wild-type and Nfix ^−/− brains were analysed. Sections from comparable positions along the rostrocaudal axis were imaged using an upright microscope (Zeiss upright Axio-Imager Z1) fitted with an Axio-Cam HRc camera. For counts of BBX-positive cells within the ventricular zone of the neocortex and hippocampus, the number of immunopositive cells per 100 μm of ventricular zone was quantified using images of immunohistochemically labelled paraffin sections at E16. For all experiments, data represent pooled results from at least 5 wild-type and 5 Nfix ^−/− brains. Counts were performed blind to the genotype of the sample. Statistical analyses were performed using a two-tailed unpaired t test. Error bars represent the standard error of the mean.

Immunofluorescence Staining

Immunofluorescence staining was performed on 6 μm paraffin sections using the BBX antibody (1/500) and a mouse-anti-nestin antibody (Developmental Studies Hybridoma Bank, rat-401, 1/100). The secondary antibodies used were a goat-anti mouse IgG Alexa 488 and a goat-anti-rabbit IgG Alexa 594 (Invitrogen; both 1/500). Images were obtained using an upright microscope (Zeiss upright Axio-Imager Z1).

Bioinformatic Promoter Screen

The NFI-binding motif was generated as reported previously (Heng et al. 2013) from published chromatin immunoprecipitation-sequencing (ChIP-seq) data for NFI (pan-NFI antibody used) (Pjanic et al. 2011). The DNA-binding domains of all NFI proteins are highly similar (Mason et al. 2009). In brief, we performed motif discovery using the MEME algorithm (Bailey et al. 2009) on ChIP-seq peaks redeclared using the ChIP-Peak algorithm (Schmid and Bucher 2010) from the published ChIP-seq ‘tag’ data for NFI. We then identified potential NFI-binding sites by scanning the complete mouse genome downloaded from the UCSC Genome Browser (mm9, July 2007) (Fujita et al. 2011) using the MEME-derived motif and the FIMO motif-scanning program (Grant et al. 2011). FIMO was run on the mouse genome (without repeat masking) using a 0-order background, generated on the entire mouse genome, and a pseudocount of 0.1. All potential binding sites with p value ≤ 10⁻⁴ were reported. The putative NFI-binding sites near the promoter of Bbx (chr16:50437453-50437467) were identified manually by viewing the FIMO output using the UCSC genome browser. We identified two potential sites upstream of the transcription start site (−4951 and −5758 relative to the transcription start site) and one downstream of the transcription start site (+222). We focussed on the first site upstream of the transcription start site for further analysis. To search for other potential transcription factor binding sites within the Bbx promoter, we searched the region around the transcription start site of Bbx (−6000 to +1000 base pairs) using FIMO and the SELEX motifs from Jolma et al. 2013, the UniPROBE mouse motifs (Newburger and Bulyk 2009) and the JASPAR vertebrate motifs (Sandelin et al. 2004). Collectively, these data provide a total of 1271 potential motifs. We identified 2692 predicted sites within this region at the (unadjusted) p value threshold of 10⁻⁴. To refine this list, we narrowed our screen to sites with FIMO p < 10⁻⁶ that were within 250 base pairs either side of the putative NFI site located at −4951. This revealed two additional motifs, namely, for the proteins Gm397 (−4754) and ZFP281 (−5208).

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assays were performed using radiolabeled annealed oligonucleotides as described previously (Heng et al. 2013). Nuclear extracts were isolated from COS cells expressing an HA-tagged NFIX expression construct or an HA-tagged AP2 expression construct. Oligonucleotide sequences were as follows: Bbx upper—5′-CCGGGTGAGTGAGGCCTCCTTGGGCTCTTCCCCATCCACATTAACTTGTTC-3′; Bbx lower 5′-CCGGGAACAAGTTAATGTGGATGGGGAAGAGCCCAAGGAGGCCTCACTCAC-3′.

Luciferase Assay

The constructs used in the luciferase assay were an NFIX expression construct and a construct containing 942 base pairs of the upstream promoter of Bbx (from −4426 to −5368 relative to the transcription start site) that included the putative NFI-binding site at −4951 cloned into the pGL4.23 vector (Promega). DNA was transfected into Neuro2A cells. A Renilla luciferase vector was added to each transfection as a normalisation control. After 48 h, luciferase activity was measured using a dual luciferase system (Promega). Within each experiment, each treatment was replicated 6 times. Each experiment was also independently replicated a minimum of three times. Statistical analyses were performed using an ANOVA with a post hoc Tukey’s test. Error bars indicate the standard error of the mean.

Results

Microarray analysis performed on the hippocampus of littermate E16 Nfix ^+/+ and Nfix ^−/− mice has previously identified over 1000 genes as being differentially expressed within the mutant, with over 65 transcription factors being upregulated (Heng et al. 2013). To determine which of these may be putative NFIX targets as assessed by being expressed in the same brain regions as NFIX, we first interrogated the mRNA expression profiles of these genes within the Allen Brain Atlas. One gene whose expression pattern within the nascent forebrain was highly specific was the transcription factor Bbx, a member of the SOX (Sry-related HMG box) family. Bbx showed a specific pattern of expression within the emerging forebrain, being expressed by progenitor cells within the ventricular zone of the embryonic neocortex and hippocampus (http://developingmouse.brain-map.org, search reference Bbx) at E15.5. To more clearly delineate the expression of this factor in the developing forebrain, we analysed the expression of BBX in the forebrain at a protein level using immunohistochemistry on 6 μm paraffin sections. At E14, expression of BBX was clearly evident within the nuclei of cells in the neocortical and hippocampal ventricular zone (Fig. 1 a–e). Furthermore, co-immunofluorescence staining with the neural progenitor cell marker nestin revealed that BBX-expressing nuclei within the ventricular zone were surrounded by nestin-positive fibres, suggesting that progenitor cells within the ventricular zone do indeed express BBX (Fig.1 f–h). At E16, expression of BBX within the hippocampus was more widespread, with ventricular zone progenitor cells and cells within the subventricular zone and intermediate zone expressing this transcription factor. Moreover, cells within the dentate migratory stream were also immunoreactive for BBX (Fig. 1 i–l). Together, these findings suggest that BBX plays a role in forebrain development.

Fig. 1.

Expression of BBX in the developing hippocampus. a–e Coronal paraffin section (6 μm) of an E14 brain showing the expression of BBX via immunohistochemistry (IHC). BBX was strongly expressed within the ventricular zone of the neocortex (arrowhead in a) and hippocampus (arrow in a). b BBX is expressed by cells within the ventricular zone of the hippocampus (arrow in b). c is a magnified view of the boxed area in b, revealing the nuclear localisation of BBX within ventricular zone cells of the hippocampus. d BBX is also expressed by ventricular zone cells within the neocortex. e At E14, there is no immunoreactivity for BBX within the developing thalamus. f–h Co-immunofluorescence staining of a coronal 6 μm paraffin section reveals that BBX-expressing nuclei within the hippocampal ventricular zone (f) are surrounded by nestin-expressing fibres (g), suggesting that neural progenitor cells express BBX (arrowheads in f, h). i–l Coronal paraffin section (6 μm) of an E16 brain showing the expression of BBX (i). BBX is expressed not only by cells within the neocortical ventricular zone (nVZ; arrowheads in j) and the hippocampal ventricular zone (hVZ; arrows in j) at this age, but also by cells within the dentate migratory stream (arrows in k) and by cells within the subventricular zone and intermediate zone (arrows in l). Scale bar (in l): a 600 μm; b 300 μm; c–e 50 μm; f–h 25 μm; i 250 μm; j–l 75 μm

We next investigated the expression of BBX within the cortex of E16 Nfix ^−/− mice. In line with our previous microarray data, we observed using 50 μm coronal brain sections that the expression of BBX within the ventricular zone of the neocortex and hippocampus of Nfix ^−/− mice was elevated when compared to littermate controls (Fig. 2 a, b). To quantify this, we performed immunohistochemistry against BBX on 6 μm paraffin sections and counted the number of immunopositive cells within the ventricular zone of the neocortex and hippocampus of wild-type and mutant brains (Fig. 2 c–f). We found significantly more neocortical and hippocampal ventricular zone cells expressing BBX within the mutant (Fig. 2 g), suggesting that this transcription factor is a target for transcriptional repression by NFIX during development.

Fig. 2.

BBX expression is upregulated in the forebrain of Nfix ^−/− mice at E16. a, b Coronal vibratome sections (50 μm) of E16 wild-type and Nfix ^−/− brains, showing the expression of BBX. There were more cells in the hippocampal and neocortical ventricular zone of the mutant expressing BBX (arrows in b) than within the control (arrowheads in a). Coronal paraffin sections (6 μm) of wild-type (c, e) and Nfix mutant (d, f) brains processed for immunohistochemistry against BBX. There were significantly more cells expressing BBX within the neocortical and hippocampal ventricular zone of the mutant (arrows in d, f) than the control (arrowheads in c, e) at this age (g). VZ ventricular zone. ** p < 0.01, t-test. Scale bar (in f): a, b 600 μm; c–f 75 μm

To ascertain whether Bbx was a direct target for transcriptional regulation by NFIX, we next performed an in silico bioinformatic screen of the Bbx promoter, looking for putative NFI-binding sites. This revealed three potential NFI-binding sites near the Bbx transcription start site (Fig. 3 a). We focussed on the first site upstream of the transcription start site, as this was predicted in silico to have a high probability of being a bona fide binding site (Fig. 3 b). Moreover, scanning around this putative NFI site also identified potential DNA-binding motifs for the transcription factors Gm397 and ZFP281 (Fig. 3 a). The latter has been implicated in controlling the pluripotency of embryonic stem cells (Wang et al. 2008), suggestive of this region of the Bbx promoter being important for the expression of Bbx in vivo. To assess the functional relevance of the predicted NFI site at −4951, we performed electrophoretic mobility shift assays using nuclear extracts from NFIX-expressing COS cells. Oligonucleotide probes encompassing the genomic region containing the putative NFI site at −4951 were bound by a molecule within the nuclear extract (Fig. 3 c). Using an anti-HA antibody (the NFIX expression construct carries a HA-tag), we further demonstrated a decrease in the mobility of the bound oligonucleotide in a supershift assay (Fig. 3 c), suggesting that NFIX can directly bind to this site within the Bbx promoter. Finally, we performed a reporter gene assay, using a luciferase construct under the regulatory control of a 942 base pair fragment of the Bbx promoter that included the NFI motif at −4951. NFIX was able to repress luciferase expression controlled by the Bbx promoter fragment (Fig. 3 d). Collectively, these data suggest that NFIX can repress the transcription of Bbx.

Fig. 3.

NFIX can repress Bbx promoter-driven transcriptional activity. a Schematic of the Bbx promoter, showing the three predicted NFI-binding sites reported by FIMO. We focussed on the first site upstream of the transcription start site, at −4951. The zoomed in region of the Bbx promoter shown below depicts the two putative DNA-binding motifs in the vicinity of the −4951 site, namely binding motifs for Gm397 and ZFP281. Also shown is the region of the promoter used in the luciferase assay (Bbx luc construct, grey rectangle). b Sequence of the NFI-binding site reported by FIMO at −4951 base pairs upstream of the Bbx transcription start site. The NFI-binding motif used within the genome-wide scan is also shown. c Nuclear extracts from COS cells expressing an HA-tagged AP2 construct (lanes 1 and 2) or an HA-tagged NFIX construct (lanes 3 and 4) were used in an electrophoretic mobility shift assay. The extracts were incubated with a radiolabeled probe containing the −4951 site. While the non-specific transcription factor AP2 did not demonstrate any specific binding to the oligonucleotide probe, the nuclear extract from the NFIX-expressing cells exhibited significant binding to the probe (asterisk, lane 3). Moreover, the addition of an anti-HA antibody to the binding reaction depleted this complex and produced a supershifted complex (SS; lane 4). FP free probe. d Transfection of Neuro2a cells with an Nfix expression vector (Nfix pCAGIG) elicited no luciferase activity in a reporter gene assay, whereas transfection of a luciferase construct under the control of the Bbx promoter element elicited robust induction of the reporter gene. Co-transfection of Nfix with the Bbx promoter reporter yielded a significantly reduced level of luciferase activity. ** p < 0.01, ANOVA

Discussion

NFIX has been shown to regulate multiple aspects of nervous system development, including repressing progenitor-specific genes such as Sox9 during embryonic cortical development (Heng et al. 2013) and activating the expression of glial-specific genes such as Gfap, brain fatty acid binding protein and Ykl-40 within cultured cells in vitro (Brun et al. 2009; Singh et al. 2011). Here, we demonstrate that NFIX also regulates the expression of the novel transcription factor Bbx during cortical development, revealing another facet of the program through which NFIX modulates corticogenesis.

BBX is strongly expressed by ventricular zone neural progenitor cells within the cortex, and significantly more cells expressing this transcription factor are found in the ventricular zone of Nfix ^−/− mice. An alternative explanation for the phenotype observed here is that the increase in cell numbers is the result of post-mitotic neurons failing to migrate away from the ventricular zone. However, we consider this unlikely, as our studies on different Nfi mutant strains have revealed that a key role for these transcription factors is to promote neural progenitor cell differentiation within the forebrain and that in their absence, the production of post-mitotic progeny is significantly delayed (Barry et al. 2008; Piper et al. 2010). For example, we have recently shown that neural progenitor cell differentiation is delayed within the hippocampus and neocortex of Nfix ^−/− mice at E16, with resulting delays in forebrain neurogenesis being evident (Heng et al. 2013). Rather, our findings here point to BBX-expressing ventricular zone progenitor cells being more prevalent in the absence of NFIX. As NFIX is postulated to drive progenitor cell differentiation within the embryonic forebrain (Heng et al. 2013), these data suggest that BBX may promote progenitor cell self-renewal. However, the means by which BBX functions in this regard is currently unclear. BBX is a member of the SOX family of transcription factors, many of which have been implicated in neural progenitor cell self-renewal (Bani-Yaghoub et al. 2006; Scott et al. 2010), indicating that BBX may play a functionally analogous role developmentally. Moreover, BBX has been reported to bind DNA and to promote the G1/S phase transition in yeast (Sanchez-Diaz et al. 2001), providing further evidence of a role for this transcription factor in cell cycle progression.

Interestingly, Bbx knockout mice have been generated by the European Conditional Mouse Mutagenesis Program (Friedel et al. 2007). Although these mice exhibit some phenotypic abnormalities, such as altered heart weight, analysis of the nervous system of postnatal knockout mice (at 6 and 16 weeks) did not reveal any gross morphological abnormalities using cresyl violet staining (http://www.sanger.ac.uk/mouseportal/phenotyping/MAEZ/mp-report/nervous-system/). These findings do not, however, rule out a role for BBX during forebrain development, and future studies aimed at analysing cortical neural progenitor cell self-renewal and their subsequent neuronal and glial differentiation during the period of corticogenesis may reveal deficits that will help elucidate the role of this transcription factor within the embryonic brain.