Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib-deficient mice
- Michael Piper1,
- Randal X Moldrich1,
- Charlotta Lindwall1, 2,
- Erica Little1,
- Guy Barry1,
- Sharon Mason1,
- Nana Sunn1,
- Nyoman Dana Kurniawan3,
- Richard M Gronostajski4 and
- Linda J Richards1, 5, 6Email author
© Piper et al. 2009
Received: 5 August 2009
Accepted: 4 December 2009
Published: 4 December 2009
Agenesis of the corpus callosum is associated with many human developmental syndromes. Key mechanisms regulating callosal formation include the guidance of axons arising from pioneering neurons in the cingulate cortex and the development of cortical midline glial populations, but their molecular regulation remains poorly characterised. Recent data have shown that mice lacking the transcription factor Nfib exhibit callosal agenesis, yet neocortical callosal neurons express only low levels of Nfib. Therefore, we investigate here how Nfib functions to regulate non-cell-autonomous mechanisms of callosal formation.
Our investigations confirmed a reduction in glial cells at the midline in Nfib -/- mice. To determine how this occurs, we examined radial progenitors at the cortical midline and found that they were specified correctly in Nfib mutant mice, but did not differentiate into mature glia. Cellular proliferation and apoptosis occurred normally at the midline of Nfib mutant mice, indicating that the decrease in midline glia observed was due to deficits in differentiation rather than proliferation or apoptosis. Next we investigated the development of callosal pioneering axons in Nfib -/- mice. Using retrograde tracer labelling, we found that Nfib is expressed in cingulate neurons and hence may regulate their development. In Nfib -/- mice, neuropilin 1-positive axons fail to cross the midline and expression of neuropilin 1 is diminished. Tract tracing and immunohistochemistry further revealed that, in late gestation, a minor population of neocortical axons does cross the midline in Nfib mutants on a C57Bl/6J background, forming a rudimentary corpus callosum. Finally, the development of other forebrain commissures in Nfib-deficient mice is also aberrant.
The formation of the corpus callosum is severely delayed in the absence of Nfib, despite Nfib not being highly expressed in neocortical callosal neurons. Our results indicate that in addition to regulating the development of midline glial populations, Nfib also regulates the expression of neuropilin 1 within the cingulate cortex. Collectively, these data indicate that defects in midline glia and cingulate cortex neurons are associated with the callosal dysgenesis seen in Nfib-deficient mice, and provide insight into how the development of these cellular populations is controlled at a molecular level.
Deleted in colorectal cancer
diffusion tensor magnetic resonance imaging
glial fibrillary acidic protein
astrocyte-specific glutamate transporter
quantitative real-time PCR.
Axonal fibre tracts enable the transfer of information between discrete parts of the brain. Within the cerebral cortex, the corpus callosum (CC), which comprises the largest fibre tract in the brain, provides connectivity between the left and right cerebral hemispheres [1, 2]. The flow of information facilitated by this tract plays an integral role in many critical functions, including behaviour, emotion and higher order cognition. Indeed, defective development of this tract in humans is correlated with a large number of syndromes, such as Mowat Wilson syndrome and Aicardi syndrome, as well as disorders including autism and schizophrenia . Formation of the CC requires a series of dynamic events to be co-ordinated both spatially and temporally during both embryogenesis and the postnatal period. These include correct patterning of the midline, differentiation and specification of callosal neurons within the nascent cortical plate, the development of distinct midline glial populations, targeting of axons to the contralateral hemisphere and the elimination of those supernumerary axons overproduced during development [1, 4]. However, while the clinical significance of the CC has long been known, our understanding of the molecular determinants underlying formation of this fibre tract remains incomplete.
Research has begun to identify some of the molecular components regulating different aspects of callosal formation. For instance, the DNA-binding protein Satb2 was recently implicated as a key determinant controlling the specification of callosally projecting neurons within the cortex [5, 6]. Furthermore, axon guidance cues, including Netrin 1 , class III semaphorins  and Slit2 , as well as guidance receptors such as DCC , neuropilin 1 (Npn1) , Robo1  and Ryk  have been implicated in callosal development. In addition, the activity of callosal neurons during development is known to be required for axonal targeting and specificity through pruning within the contralateral hemisphere [4, 14].
Another critical determinant of CC formation is the development of distinct glial populations at the cortical midline . Two midline glial populations, the glial wedge and the indusium griseum glia, are believed to regulate callosal development, in part through expression of guidance cues such as Slit2 [16, 17]. Given that these glia develop relatively early compared to other cortical glial populations , identifying the factors regulating their development is important for understanding both glial development and axonal guidance. One gene family in particular, the Nuclear Factor One (Nfi) transcription factors, has been shown to play a central role in regulating glial development and axon tract formation during embryogenesis. Nfia was recently implicated in regulating gliogenesis in the spinal cord , and Nfia-deficient mice have been demonstrated to have severely reduced glial formation at the cortical midline . Similarly, Nfib has been shown to regulate the formation of glia within the ammonic neuroepithelium of the developing hippocampus .
Mice lacking Nfib have glial defects at the midline, as well as agenesis of the CC , but whether these defects are mechanistically related is unknown. Here we demonstrate that neither excessive apoptosis, nor aberrant proliferation at the midline, underlies the glial defect in Nfib knockout mice, but rather that radial progenitors fail to differentiate into mature glia. Furthermore, expression of Slit2 is diminished at the midline. We also report that cell-autonomous defects in cingulate cortex neurons may contribute to callosal malformation in Nfib -/- mice. Finally, we demonstrate that, at embryonic day 18 (E18), a small population of axons does cross the midline caudally in Nfib-deficient mice. These results demonstrate that Nfib is critical for the maturation of midline glia that are required for CC formation. Furthermore, defects in Npn1-expressing pioneering neurons may contribute to the callosal dysgenesis evident in Nfib -/- mice, implicating multiple non-cell-autonomous roles for Nfib in neocortical callosal formation.
NFIB is expressed at the cortical midline
Nfib-deficient mice exhibit a variety of cortical deficits, including absence of the basilar pons  and malformation of the dentate gyrus . They also exhibit agenesis of the CC at E17 as determined by staining with the axonal marker L1 . Haematoxylin staining of E18 wildtype and Nfib-deficient brains (Figure 1E-H) supported this finding, indicating that, in rostral sections of the mutant, formation of the CC was impaired.
Glial development is curtailed at the cortical midline of Nfib-deficient mice
Proliferation and cell death is normal at the cortical midline of Nfib null mutants
Radial progenitors are specified in the absence of Nfib
Expression of radial glial markers at the midline is impaired
Abnormal development of the subcallosal sling in Nfib knockout mice
Expression of Npn1 on the axons of cingulate pioneer neurons is diminished in Nfib-deficient mice
We have recently postulated that Npn1-semaphorin signalling may be vital for callosal formation . Thus, diminished expression of Npn1 on axons emanating from the cingulate cortex could also contribute to the callosal defects observed in Nfib knockout mice. To determine if NFIB was expressed in cingulate cortex neurons extending axons across the CC, we performed tract tracing using the retrograde tracer True Blue, which was injected into the cingulate cortex of E17 embryos in utero. Embryos were then perfuse-fixed at E18. Immunohistochemistry against NFIB demonstrated that some nuclei in the cingulate cortex contralateral to the injection site were both NFIB- and True Blue-positive (Figure 7G-I), indicating that a proportion of callosally projecting cingulate neurons express Nfib. Interestingly, however, not all of the projections from the cingulate cortex were abnormal in the absence of Nfib. The perforating pathway is an ipsilateral tract extending from the cingulate cortex to the medial septum/diagonal band of Broca and vice versa . Analysis of this pathway with immunohistochemistry against the axonal marker neurofilament revealed no defect, as in both wildtype and Nfib-deficient sections, neurofilament-positive axons comprising the perforating pathway intersected the callosal axons en route to their targets (Figure 7E, F).
Nfib-deficient mice exhibit callosal dysgenesis at E18
Reduced expression of Slit2 at the cortical midline of Nfib mutants
Defects in multiple forebrain commissures in Nfib mutants
The Nfi gene family is required for multiple aspects of central nervous system development . Here we show that Nfib regulates the differentiation of specific glial populations critical for formation of the CC, the glial wedge and the indusium griseum glia. In the absence of Nfib, the appearance of these glial populations is dramatically curtailed, although this is not due to aberrant apoptosis or proliferation. Rather, differentiation of these glia from radial progenitors at the cortical midline is impaired. Our data also indicate that expression of Npn1 on axons of pioneer neurons within the cingulate cortex of Nfib mutants is significantly reduced in comparison to controls. Collectively, these findings demonstrate that predominantly non-cell-autonomous defects contribute to the defects in neocortical CC formation in Nfib-deficient mice. Finally, we also show that, in late gestation, a small population of axons crosses the cortical midline at more caudal levels in Nfib mutant mice. This correlates with an increase in the levels of GFAP and Slit2, suggesting that, at least caudally, a compensatory mechanism allows both glial differentiation and CC formation in mice lacking this transcription factor.
The Nfi transcription factors are emerging as central players in glial lineage determination. Nfi genes have previously been shown to promote the expression of a suite of glial-specific genes in vitro, including those encoding GFAP , brain lipid binding protein , α1-antichymotrypsin  and tenascin C . Furthermore, a number of recent studies have corroborated these findings in vivo. For instance, Nfib has been shown to regulate glial differentiation within the ammonic neuroepithelium of the developing mouse hippocampus , while the onset of gliogenesis in the chick spinal cord requires the action of Nfia . A mechanistic insight into how Nfi genes could promote gliogenesis within cortical precursors has recently been proposed . Namihira and colleagues  demonstrated that within cortical neural progenitor cells at mid-gestation, Notch pathway signalling elicited expression of NFIA, which bound to the promoters of astrocytic genes such as that encoding GFAP, culminating in demethylation and subsequent activation of these glial genes. Our findings are readily accommodated within the conceptual framework provided by these studies, with the impairment of glial maturation at the cortical midline being a direct consequence of radial progenitors failing to differentiate in the absence of Nfib.
Our data indicate that Nfib may also contribute to callosal formation via the cell-autonomous regulation of Npn1 within neurons of the cingulate cortex. Npn1, a cognate ligand for secreted class III semaphorins, has recently been shown to contribute to the formation of the CC [8, 11, 43]. The finding that Npn1 expression is significantly downregulated in the cortex of Nfib mutants implies that the role of Nfi genes is not solely confined to gliogenesis, a result that is supported by the broad expression pattern of this gene family within neurons and glia of the developing telencephalon [21, 23]. Indeed, recent reports have highlighted significant roles for Nfi family members in neuronal development. For instance, Nfib has been shown to play a central role in precerebellar mossy fibre neuron generation within the pons , while Nfia controls axon outgrowth, dendritogenesis and migration of cerebellar granule neurons via regulation of N-cadherin and ephrin B1 .
Our finding that a rudimentary CC forms caudally in Nfib-deficient mice appears inconsistent with the initial characterisation of this knockout line, which reported agenesis of the CC . This discrepancy could lie in the age of the embryos investigated, given Steele-Perkins and colleagues conducted their analysis at E17, one day prior to when the present analysis was conducted. Furthermore, the previous study analysed the Nfib allele on a mixed genetic background (129S6/C57Bl/6J). The 129S6 strain exhibits sporadic occurrence of callosal agenesis [45, 46], which may have contributed to the complete absence of the CC observed . The delayed development of the CC we described in this study is of considerable interest, as it raises the possibility that further development of this tract may occur postnatally. Unfortunately, on the genetic background on which this strain is maintained (C57Bl/6J), Nfib knockout mice die at birth due to defective lung maturation [22, 47]. Generation of a conditional Nfib allele would be an ideal way to ablate Nfib in a cortex-specific manner, thereby enabling further investigation of CC formation postnatally in the absence of this transcription factor. Furthermore, the functional sequelae arising from delayed callosal malformation in these mice could then be investigated using behavioural analyses.
While a hypomorphic CC does form caudally at E18 in the absence of Nfib, how this is regulated remains unclear. One possibility is that the caudal CC axons in the Nfib mutant could utilise axons of the hippocampal commissure as a substrate to cross the cortical midline . Another possible determinant of CC formation in the mutant is the delayed development of midline glia within both the glial wedge and indusium griseum. Although the identities of the genes regulating the delayed glial development in the E18 mutant are unknown, other members of the Nfi gene family are excellent candidates. Nfia and Nfix are both expressed within the glial wedge and indusium griseum at late gestation , and furthermore, Nfia and Nfib are expressed in the same cells within the glial wedge at E18 (data not shown). Thus, although differentiation of radial progenitors is delayed in the absence of Nfib, compensation by other Nfi family members may provide a mechanism for mature glia to eventually form at the cortical midline, thereby enabling development of the CC. Finally, our finding that the anterior commissure and the hippocampal commissure were also disrupted in the Nfib -/- mice could indicate that similar non-cell-autonomous mechanisms, such as the development of midline glial and neuronal sling populations, may underlie the formation of all telencephalic commissures. In conclusion, our data provide a comprehensive insight into the phenotypic abnormalities underlying callosal malformation in Nfib-deficient mice, and demonstrate that multiple factors contribute to these defects during embryogenesis.
Materials and methods
Litters of wildtype C57Bl/6J and Nfib-deficient mice, bred at The University of Queensland with approval from the institutional Animal Ethics Committee, were used in this study. The Nfib -/- allele  was backcrossed for more than ten generations onto the C57Bl/6J background. Nfib +/- mice were bred to obtain wildtype, Nfib +/- and Nfib -/-progeny. No midline defects were detected in wildtype or heterozygote animals. Timed-pregnant females were obtained by placing male and female mice together overnight. The following day was designated as E0 if the female had a vaginal plug. Embryos were genotyped by PCR as previously described .
On the required gestational day, embryos were drop-fixed in 4% paraformaldehyde (PFA; E14 and below) or transcardially perfused with 0.9% phosphate buffered saline, followed by 4% PFA (E15 to E18). They were then postfixed in 4% PFA at 4°C until sectioning.
Brains of E18 wildtype C57Bl/6J or Nfib -/- embryos were dissected from the skull, blocked in 3% noble agar (Difco, Sparks, MD, USA), and then sectioned coronally at 45 μm on a vibratome (Leica, Nussloch, Germany). Sections were then mounted and stained with Mayer's haematoxylin using standard protocols.
Immunohistochemistry on floating sections
Brains were sectioned as described above, then processed free-floating for immunohistochemistry using the chromogen 3,3' diaminobenzidine as described previously . Primary antibodies used for immunohistochemistry were anti-GAP43 (mouse monoclonal, 1/100,000; Chemicon, Bedford, MA, USA), anti-GFAP (rabbit polyclonal, 1/50,000; DAKO, Glostrup, Denmark), anti-cleaved caspase 3 (rabbit polyclonal, 1/1,000; Cell Signaling Technology, Danvers, MA, USA), anti-GLAST (rabbit polyclonal, 1/50,000; a gift from Niels Danbolt, University of Oslo), anti-nestin (mouse monoclonal, 1/1,500; Developmental Studies Hybridoma Bank), anti-tenascin C (rabbit polyclonal, 1/2,000; Chemicon), anti-Tbr1 (rabbit polyclonal, 1/100,000; a gift from Robert Hevner, University of Washington), anti-NFIA (rabbit polyclonal, 1/30,000; Active Motif, Carlsbad, CA, USA), anti-Emx1 (rabbit polyclonal, 1/30,000; a gift from Giorgio Corte, The University of Genova Medical School), anti-Npn1 (rabbit polyclonal, 1/75,000; a gift from David Ginty, Johns Hopkins University) and anti-DCC (rabbit polyclonal, 1/30,000; a gift from Helen Cooper, Queensland Brain Institute). Secondary antibodies used were biotinylated goat-anti-rabbit IgG (1/1,000; Vector Laboratories, Burlingame, CA, USA) and biotinylated donkey-anti-mouse IgG (1/1,000; Jackson ImmunoResearch, West Grove, PA, USA). To perform immunofluorescent labelling, sections were incubated overnight with the primary antibody at 4°C. They were then washed and incubated in secondary antibody, before being washed again and mounted. The primary antibodies used for immunofluorescent labelling were anti-phosphohistone H3 (rabbit polyclonal, 1/1,000; Millipore, Billerica, MA, USA), anti β-galactosidase (1/1,000; Promega, Madison, WI, USA), anti-GAP43 (1/5,000), anti-DCC (1/1,000), anti-GFAP (1/2,000), anti-Satb2 (1/1,000; Abcam, Cambridge, UK) and anti-NFIB (1/1,000). The secondary antibodies used were goat-anti-rabbit IgG AlexaFluor488 and goat-anti-mouse IgG AlexaFluor594 (both 1/1,000; Invitrogen, Carlsbad, CA).
Immunohistochemistry on paraffin sections
E18 wildtype brains were perfused as above and embedded in paraffin wax. Brains were sectioned at a thickness of 6 μm. Antigen retrieval was performed using a 10 mM, pH 6 sodium citrate solution, and immunohistochemistry was performed as described above using 3,3' diaminobenzidine as the chromogen. The primary antibody used for immunohistochemistry was anti-NFIB (1/1,000, Active Motif), and a biotinylated goat-anti-rabbit IgG secondary antibody (Vector Laboratories) was used at 1/1,000.
Image acquisition and analysis
Following immunohistochemistry, sections were imaged using an upright microscope (Zeiss Z1, Zeiss, Goettingen, Germany) attached to a digital camera (Zeiss AxioCam HRc). AxioVision software (Zeiss) was used to capture images. When comparing wildtype to knockout tissue, sections from matching positions along the rostro-caudal axis were selected.
Quantification of proliferation
To quantify proliferation at the developing cortical midline, sections from E13, E14 and E15 wildtype C57Bl/6J or Nfib -/- embryos were labelled with an anti-phosphohistone H3 antibody as described above. Sections were imaged using an upright fluorescence microscope (Zeiss Z1) attached to a digital camera (Zeiss AxioCam HRc). Eight to ten optical sections encompassing the entire 45-μm section were captured with an ApoTome (Zeiss). To calculate the total number of phosphohistone H3-positive cells per unit area at the cortical midline, a 300 μm2-boxed region, encompassing the presumptive glial wedge area, was generated using AxioVision software (Zeiss). The number of immunolabelled cells in focus in each optical section of this region was counted and pooled (n = 3 for both wildtype and knockout at all ages).
For all experiments described in this study, sections from three different brains of each genotype were analysed. Statistical analyses were performed using a two-tailed unpaired t-test. Error bars represent standard error of the mean.
Carbocyanine tract tracing
E18 wildtype and Nfib -/- brains were fixed in 4% PFA as described above. A small injection of DiI (in a 10% solution of dimethylformamide; Invitrogen) was then made into the neocortex using a pulled glass pipette attached to a picospritzer. Brains were stored in the dark at 37°C in 4% PFA for at least 4 weeks to allow dye transport. They were then sectioned coronally at 45 μm using a vibratome, and imaged using an upright fluorescence microscope (Zeiss Z1). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Three brains were analysed for each genotype.
Retrograde labelling under ultrasound guidance
Pregnant mice were anaesthetised with isofluorane (2%) for the duration of the microinjection procedures. The uterine horn was exposed through an incision in the abdominal midline for the purpose of ultrasound- imaging and guided microinjections (Vevo770, VisualSonics, Toronto, Canada). Retrograde labelling of callosal axons with True Blue chloride (Invitrogen) was performed as described previously, with modifications appropriate for ultrasound-guided microinjection in utero. Embryos were visualised under a 40 MHz transducer probe (RMV711) and a small volume of the tracer (approximately 250 nL of a 1 μg/μL solution) was injected into the cortex of wildtype E17 embryos in utero through the uterine wall with the aid of a nanojector (Nanoject II, Drummond Scientific, Broomall, PA, USA). Once embryos were injected, the uterine horn was returned to the abdominal cavity, and the incision was sutured. Then, 24 hours later (E18) the embryos were perfused transcardially as described above and processed for NFIB immunohistochemistry. Fluorescence images were obtained with an upright microscope (Zeiss Z1) as described above.
In situ hybridisation
In situ hybridisation was performed as described previously , with minor modifications. An antisense riboprobe specific to Slit2 was hybridised to coronal brain sections at 65°C overnight. The colour reaction solution was BM Purple (Roche, Mannheim, Germany).
Reverse transcription and quantitative real-time PCR
The reverse transcription was performed using Superscript III (Invitrogen). Briefly, 0.5 μg total RNA was reverse transcribed with random hexamers. qPCR reactions were carried out in a Rotor-Gene 3000 (Corbett Life Science, Sydney, Australia) using the SYBR Green PCR Master Mix (Invitrogen). All the samples were diluted 1/100 with water and 5 μL of these dilutions were used for each SYBR Green PCR reaction containing 10 μL SYBR Green PCR Master Mix, 10 μM of each primer, and deionised water. The reactions were incubated for 10 minutes at 95°C followed by 40 cycles with 15 seconds denaturation at 95°C, 20 seconds annealing at 60°C, and 30 seconds extension at 72°C. Primer sequences are available on request.
Quantitative real-time PCR data expression and analysis
After completion of the PCR amplification, the data were analysed with the Rotor-Gene software (Corbett Life Science) and Microsoft Excel. In order to quantify the mRNA expression levels, the housekeeping gene HPRT was used as a relative standard. All the samples were tested in triplicate. By means of this strategy, we achieved a relative PCR kinetic of the standard and the sample. For all qPCR analyses, RNA from three independent replicates for both wildtype and Nfib mutants were interrogated. Statistical analyses were performed using a two-tailed unpaired t-test. Error bars represent the standard error of the mean.
Diffusion-weighted magnetic resonance imaging and tractography
Following perfusion fixation and phosphate-buffered saline washing, diffusion-weighted images were acquired with the samples immersed in Fomblin Y-LVAC fluid (Solvay Solexis, Italy), using a 16.4 Tesla Bruker scanner and a 10 mm quadrature birdcage coil. A three-dimensional diffusion-weighted spin-echo sequence was acquired using a repetition time of 400 ms, an echo time of 22.8 ms and an imaging resolution of 0.08 × 0.08 × 0.08 mm with a signal average of 1. Each dataset was composed of two B o and thirty direction diffusion-weighted images (b value of 5,000 s/mm2, δ/Δ = 2.5/14 ms). Reconstruction and tractography were performed with Diffusion Toolkit  according to high angular resolution diffusion (HARDI) and Q-ball models . Tractography limits were set at fractional anisotropy values greater than 0.1 and a turning angle ≤ 45°. Hippocampal commissure tractography was performed using hand-drawn regions-of-interest on colour-coded fractional anisotropy maps in TrackVis .
We thank John Baisden, Oressia Zalucki, Jane Ellis and the UQBR Animal Facility for technical assistance, Marc Tessier-Lavigne (Genentech) for the Slit2 riboprobe and Robert Hevner (University of Washington), Helen Cooper (Queensland Brain Institute), Giorgio Corte (University of Genova Medical School) and Niels Danbolt (University of Oslo) for reagents. The 16.4T facility is part of the Queensland NMR network, funded by the Queensland Government Smart State initiative. This work was funded by a National Health and Medical Research Council project grant (LJR) and a Clive and Vera Ramaciotti grant (MP). The following authors were supported by National Health and Medical Research Council fellowships: LJR (Senior Research Fellowship); MP (Howard Florey Centenary Fellowship and Biomedical Career Development Award); RXM (CJ Martin Fellowship). SM is supported by a University of Queensland F.G Meade PhD Scholarship. NDK is grateful for the support of Lembaga Eijkman, Jakarta.
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