Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib-deficient mice.

Abstract

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.

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BioMed Central
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Neural Development
Open Access
Research article
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 Richards*1,5,6
Address: 1Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia, 2Institute for Neuroscience and Physiology,
Sahlgrenska Academy, University of Gothenburg, Sweden, 3Centre for Magnetic Resonance, The University of Queensland, Brisbane, Queensland,
Australia, 4Department of Biochemistry and the Program in Neuroscience, State University of New York at Buffalo, Buffalo, NY, USA, 5Center of
Excellence in Bioinformatics and Life Sciences, Buffalo, NY, USA and 6The School of Biomedical Sciences, The University of Queensland, Brisbane,
Queensland, Australia
Email: Michael Piper - m.piper@uq.edu.au; Randal X Moldrich - r.moldrich@uq.edu.au; Charlotta Lindwall - charlotta.lindwall@neuro.gu.se;
Erica Little - e.little@uq.edu.au; Guy Barry - g.barry@uq.edu.au; Sharon Mason - s.mason1@uq.edu.au; Nana Sunn - n.sunn@uq.edu.au;
Nyoman Dana Kurniawan - n.kurniawan@uq.edu.au; Richard M Gronostajski - gronos@mac.com; Linda J Richards* - richards@uq.edu.au
* Corresponding author
Abstract
Background: 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.
Results: 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.
Conclusion: 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.
Published: 4 December 2009
Neural Development 2009, 4:43doi:10.1186/1749-8104-4-43
Received: 5 August 2009
Accepted: 4 December 2009
This article is available from: http://www.neuraldevelopment.com/content/4/1/43
© 2009 Piper et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
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 inte-
gral 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 syn-
drome and Aicardi syndrome, as well as disorders includ-
ing autism and schizophrenia [3]. 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 develop-
ment 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 signifi-
cance 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 forma-
tion. 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 [7], class III semaphorins [8] and Slit2 [9], as well
as guidance receptors such as DCC [10], neuropilin 1
(Npn1) [11], Robo1 [12] and Ryk [13] have been impli-
cated in callosal development. In addition, the activity of
callosal neurons during development is known to be
required for axonal targeting and specificity through prun-
ing within the contralateral hemisphere [4,14].
Another critical determinant of CC formation is the devel-
opment of distinct glial populations at the cortical mid-
line [15]. Two midline glial populations, the glial wedge
and the indusium griseum glia, are believed to regulate
callosal development, in part through expression of guid-
ance cues such as Slit2 [16,17]. Given that these glia
develop relatively early compared to other cortical glial
populations [18], 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 fac-
tors, 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 [19], and Nfia-deficient
mice have been demonstrated to have severely reduced
glial formation at the cortical midline [20]. Similarly, Nfib
has been shown to regulate the formation of glia within
the ammonic neuroepithelium of the developing hippoc-
ampus [21].
Mice lacking Nfib have glial defects at the midline, as well
as agenesis of the CC [22], but whether these defects are
mechanistically related is unknown. Here we demonstrate
that neither excessive apoptosis, nor aberrant prolifera-
tion 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 con-
tribute 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-express-
ing pioneering neurons may contribute to the callosal dys-
genesis evident in Nfib-/- mice, implicating multiple non-
cell-autonomous roles for Nfib in neocortical callosal for-
mation.
Results
NFIB is expressed at the cortical midline
Although we previously showed by retrograde labelling
that NFIB was not highly expressed in neocortical callosal
neurons [23], we wanted to examine what other cells
expressed NFIB at the midline. Immunohistochemistry on
6 μm coronal paraffin sections from E18 wildtype brains
(Figure 1A) demonstrated the expression of NFIB within
the glial wedge and the indusium griseum, two glial pop-
ulations essential for development of the CC (Figure 1B).
Expression was also detected in the subcallosal sling (Fig-
ure 1B), a stream of cells originating in the subventricular
zone that crosses the cortical midline ventral to the devel-
oping CC [24,25]. The Nfib knockout line was generated
with a nuclear-localised lacZ reporter gene [22]. Expres-
sion of β-galactosidase at the cortical midline in E18 het-
erozygote brains was co-incident with that of NFIB
(Additional file 1), enabling us to use the expression of β-
galactosidase as a reliable indicator of those cells express-
ing NFIB. To determine if the cells in the glial wedge and
indusium griseum expressing Nfib were in fact glia, we
double labelled E18 coronal sections with antibodies
against β-galactosidase (a mouse monoclonal antibody)
and glial fibrillary acidic protein (GFAP; a rabbit polyclo-
nal antibody), a marker for mature glial cells. Many β-
galactosidase-positive cells in both the glial wedge and the
indusium griseum were surrounded by GFAP-positive
fibres, indicating that these glial populations are likely to
express Nfib (Figure 1C, D).

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Nfib-deficient mice exhibit a variety of cortical deficits,
including absence of the basilar pons [26] and malforma-
tion of the dentate gyrus [21]. They also exhibit agenesis
of the CC at E17 as determined by staining with the
axonal marker L1 [22]. Haematoxylin staining of E18
wildtype and Nfib-deficient brains (Figure 1E-H) sup-
ported 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
The development of mature, GFAP-expressing glial popu-
lations at the cortical midline of Nfib-/- mice at E17 was
reported to be reduced in the absence of this transcription
factor [22]. To gain a deeper insight into glial develop-
ment during callosal formation, we first conducted a time-
course of GFAP expression in Nfib-deficient and littermate
control brains (Figure 2). In sections from wildtype mice,
GFAP-expressing cells were first seen in the glial wedge at
E15, and within the indusium griseum glia and a third
midline glial population, the midline zipper glia, at E17
(Figure 2A, C, E). In the mutant, however, GFAP expres-
sion at the cortical midline was delayed until E17, when a
small population of GFAP-expressing cells became evi-
dent within the glial wedge (Figure 2B, D, F). By E18,
expression of GFAP within the glial wedge in the mutant
had increased, but was still reduced in comparison to that
seen in wildtype controls (Figure 2G, H). Moreover, at this
rostro-caudal position, no GFAP expression was observed
in the indusium griseum or in the midline zipper glia of
the mutant mice. As the indusium griseum contains both
glia and neurons [27], we also examined the expression of
Tbr1, a marker of post-mitotic neurons. These data
revealed that neurons were still present in the indusium
griseum of mice lacking Nfib (Figure 2J), from which we
infer that development of this structure per se is not
affected. Rather, it is specifically the development of mid-
line glial populations that is aberrant in the Nfib mutants.
Proliferation and cell death is normal at the cortical
midline of Nfib null mutants
The absence of cortical midline glial cells within Nfib-defi-
cient mice may arise from deficiencies in cellular prolifer-
ation at the midline, excessive cellular apoptosis, or
through a failure of cortical progenitors to differentiate
into mature glia during embryogenesis. To determine
which of these processes was responsible for the defect in
glial development we observed in Nfib null mutants, we
first investigated cellular proliferation at the cortical mid-
line. We have demonstrated previously that glia within
the glial wedge and indusium griseum are born predomi-
nantly between E13 and E15 [18]. We therefore analysed
proliferation at the cortical midline at these ages in both
wildtype and Nfib-deficient brains using the mitotic
marker phosphohistone H3 (PH3; Figure 3A, B). As these
Expression of NFIB in midline glial populations
Figure 1
Expression of NFIB in midline glial populations. (A, B)
Coronal section of an E18 wildtype brain stained with NFIB.
(A) NFIB was expressed broadly throughout the dorsal telen-
cephalon (dTel). (B) Higher magnification view of the boxed
region in (A) showing NFIB expression at the cortical midline.
NFIB was expressed in the glial wedge (GW), indusium gri-
seum glia (IGG) and subcallosal sling (SS). (C, D) Confocal
sections of E18 Nfib heterozygote brains, demonstrating the
co-expression of the β-galactosidase (β-gal) reporter (red)
and glial fibrillary acidic protein (GFAP; green) within the glial
wedge (C) and the indusium griseum glia (D). β-gal-positive
nuclei were often surrounded by GFAP-positive fibres
(arrows in C, D), indicating that GFAP-expressing glia likely
express Nfib. (E-H) Coronal sections of wildtype (E, G) and
Nfib knockout (F, H) brains stained with haematoxylin. The
corpus callosum (CC) does not form rostrally in mice lacking
Nfib (arrow in (H)). Panels (G) and (H) are higher magnifica-
tions of the boxed regions in (E) and (F), respectively. Scale
bars: 500 μm (A, E, F); 200 μm (B, G, H); 100 μm (C, D).

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data revealed no significant difference in the number of
PH3-positive cells between Nfib mutant and wildtype sec-
tions at the cortical midline at E13, E14 or E15 (Figure
3C), we next investigated cell death using the apoptotic
marker cleaved caspase 3. Very few cleaved caspase 3-pos-
itive cells were seen at the cortical midline of either
wildtype or Nfib-deficient brains, and those that were
found were located almost exclusively where the cerebral
hemispheres fuse (arrows in Figure 3D, E). Furthermore,
we observed no significant differences in apoptotic cell
death at the midline between Nfib mutants and controls
between E14 and E18 (Figure 3F). Collectively, these data
indicate that neither aberrant proliferation nor excessive
apoptosis are responsible for the phenotypic abnormali-
ties observed in the absence of Nfib. We therefore next
examined the specification of radial progenitor cells in
Nfib-deficient brains, and their capacity to differentiate
into mature glia.
Radial progenitors are specified in the absence of Nfib
The intermediate filament protein nestin is expressed
from E10.5 onwards in radial progenitors within the
developing cortical neuroepithelium [28], and as such,
nestin expression can be used to monitor the specification
of radial progenitors within the cortex during embryogen-
esis [29]. Nestin expression at the cortical midline of Nfib-
deficient mice between E14 and E18 was grossly normal
when compared to that of littermate controls (Figure 4).
However, analysis of nestin mRNA at E18 using quantita-
tive real-time PCR (qPCR) on wildtype and Nfib-deficient
cortical tissue showed that there were significantly higher
levels of nestin in the Nfib mutant compared to wildtype
controls at this age (P < 0.05, t-test; Figure 4G), indicative
of a retention of progenitor cells in the mutant. These
findings suggest that radial progenitors are specified in the
absence of this transcription factor, yet are delayed in their
differentiation to GFAP-positive glia in late gestation.
Expression of radial glial markers at the midline is
impaired
As nestin-expressing radial progenitors differentiate into
radial glia, they begin expressing astroglial markers. Two
glial-specific markers used previously to investigate the
differentiation of radial progenitors in the cortex are astro-
cyte-specific glutamate transporter (GLAST) [30] and the
extracellular matrix molecule tenascin C [31]. To deter-
mine whether the lack of GFAP-positive midline glia in
Nfib-deficient mice was due to a defect in the differentia-
tion of radial progenitors, we analysed the expression of
these markers during development. At E14 in wildtype
mice, GLAST expression was observed in the ventricular
zone of the cortex, but was higher within the region of the
presumptive glial wedge (Figure 5A). This expression pat-
tern was even more evident in the wild type at E16 (Figure
5C). However, in mutant mice, expression of GLAST was
Reduced expression of GFAP in Nfib knockout mice
Figure 2
Reduced expression of GFAP in Nfib knockout mice.
(A-H) Expression of GFAP at the cortical midline of wildtype
(A, C, E, G) and Nfib-deficient (B, D, F, H) mice. In the
wildtype, expression of GFAP in the glial wedge was initiated
at E15, and became progressively stronger as development
proceeded (arrows in (A, C, E, G)). GFAP expression in the
indusium griseum glia (arrowhead) and midline zipper glia
(double arrowhead) of the wildtype was also evident from
E17 onwards (E, G). However, in the mutant, low levels of
GFAP expression in the glial wedge were only observed from
E17 onwards (open arrowheads in (F, H)), whereas expres-
sion in the indusium griseum glia and midline zipper glia was
absent. Neurons in the indusium griseum of the wildtype
expressed Tbr1 (arrows in (I)). Neurons expressing Tbr1 in
the indusium griseum were also observed in the mutant
(arrows in (J)). Scale bars: 280 μm (A, B); 250 μm (C, D); 225
μm (E, F); 200 μm (G-J).

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clearly diminished in comparison to wildtype controls at
both E14 and E16 (Figure 5B, D). At E18 in wildtype mice,
GLAST expression was observed in the glial wedge, the
indusium griseum glia and the midline zipper glia (Figure
5E). In mutant mice, GLAST immunoreactivity in the glial
wedge was diminished, and moreover, this marker, like
GFAP (Figure 2), was detected in neither the indusium gri-
seum glia nor the midline zipper glia (Figure 5F), indicat-
ing that these mature glial populations appear absent in
Nfib-deficient mice. Expression of tenascin C was also
compromised in mice lacking Nfib. Comparison of
wildtype and Nfib-deficient brains at E15 and E16 indi-
cated that levels of this extracellular matrix molecule were
higher in the wildtype than in the mutant (Additional file
2). Taken together, these data indicate that glial develop-
ment is indeed impaired in Nfib knockout mice, and sug-
gest that the lack of mature, GFAP-expressing glia at the
cortical midline of the mutant results from aberrant differ-
entiation of radial progenitors.
Abnormal development of the subcallosal sling in Nfib
knockout mice
The subcallosal sling is a population of neurons that
migrate from the medial aspect of the lateral ventricles
beneath the nascent CC. Migration of these neurons
begins at approximately E15 and continues postnatally
[24]. To determine whether formation of this structure
was perturbed in mice lacking Nfib, we performed immu-
nohistochemistry using two markers expressed by sling
neurons, NFIA and Emx1 [24]. In wildtype mice at E18,
sling neurons were observed beneath the CC (Figure 6A,
C). In Nfib mutant mice, however, sling neurons were
unable to cross the midline, perhaps due to the failure of
the CC to form. Unlike in Nfia-deficient mice, where sling
cells invade the septum [20], these cells appear to stall
near the midline in the absence of Nfib and do not invade
the septum (Figure 6B, D). This phenotype was also seen
with a third marker for the subcallosal sling, calretinin
(data not shown). These data suggest that correct forma-
tion of the sling requires Nfib function, and further indi-
cates that Nfia and Nfib may play different roles in relation
to subcallosal sling formation.
Expression of Npn1 on the axons of cingulate pioneer
neurons is diminished in Nfib-deficient mice
The failure of the glial wedge and the indusium griseum
glia to form may underlie the callosal defects previously
described for Nfib-deficient mice [22], as these glial popu-
lations have been postulated to be pivotal for the forma-
tion of this axonal tract [16,17]. We next investigated
Normal proliferation and cell death at the cortical midline of mice lacking Nfib
Figure 3
Normal proliferation and cell death at the cortical midline of mice lacking Nfib. (A, B) Proliferation at the cortical
midline in wildtype (A) and Nfib-deficient (B) mice was assessed with immunohistochemistry against the mitotic marker phos-
phohistone H3. (C) Counts of phosphohistone H3-positive cells at the cortical midline demonstrated that there was no signif-
icant difference in proliferation between Nfib null mutants and controls at E13, E14 or E15. (D, E) Apoptosis at the midline in
wildtype (D) and Nfib-deficient (E) brains was assessed via expression of the marker for cell death, cleaved caspase 3. There
were few apoptotic cells observed in either wildtype or knockout samples (arrows in (D, E)), and these were predominantly
observed around the area where fusion between the cerebral hemispheres occurs. (F) We did not observe any significant dif-
ferences in the numbers of apoptotic cells in mice lacking Nfib compared to wildtype controls at E14, E15 or E18. n = 3 inde-
pendent replicates for both wildtype and Nfib mutants. Error bars indicate standard error of the mean. Scale bar: 300 μm.

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whether defects in callosally projecting neurons could
contribute to the CC defects observed in Nfib-/- mice. Two
populations of neurons extend axons that cross the mid-
line via the CC; neocortical callosal neurons and cingulate
cortex callosal neurons [1]. The latter are thought to be
critical for pioneering callosal tract formation, as neurons
from the cingulate cortex are the first to extend axons
across the CC [32,33]. Interestingly, retrograde labelling
in early postnatal (P5) wildtype mice has previously
shown that most callosally projecting neocortical neurons
do not express NFIB at this age [23]. However, the broad
expression pattern of NFIB in the cingulate cortex at E18
(Figure 1A) suggests that, in addition to glial deficits, cell-
autonomous neuronal defects within the cingulate cortex
may contribute to the callosal phenotype in embryonic
Nfib-deficient mice. To investigate this, we analysed pro-
jections arising from the cingulate cortex. Deleted in
colorectal cancer (DCC) is a marker expressed selectively
on callosal axons arising from neurons within the cingu-
late cortex [34]. In wild types at E18, DCC-expressing
axons could be seen crossing the midline in the dorsal
region of the CC (Figure 7A). In the mutant, DCC-express-
ing axons extended towards the midline (Figure 7B) but
failed to cross. These data indicate that neurons within the
cingulate cortex were indeed able to extend axons towards
the presumptive CC, and that this was not a growth defect.
However, expression of another guidance receptor specif-
Radial progenitor cells express nestin at higher levels at E18 in Nfib mutant mice
Figure 4
Radial progenitor cells express nestin at higher lev-
els at E18 in Nfib mutant mice. (A-F) Coronal sec-
tions of wildtype (A, C, E) and Nfib-deficient brains (B, D,
F) demonstrating expression of nestin. At E14 (A, B), E16
(C, D) and E18 (E, F), expression of nestin in the mutant
was comparable to that in the control. (G) At E18, levels
of nestin mRNA were significantly higher in Nfib mutants
than in littermate controls (*P < 0.05; t-test). RNA from
three independent replicates for both wildtype (WT) and
Nfib mutants (Nfib knockout (KO)) was analysed. Error
bars indicate standard error of the mean. Scale bar: 300
μm (A, B); 250 μm (C, D); 200 μm (E, F).
Diminished expression of GLAST at the cortical midline of
Nfib-deficient mice
Figure 5
Diminished expression of GLAST at the cortical mid-
line of Nfib-deficient mice. (A-F) Expression of GLAST
at E14 (A, B), E16 (C, D) and E18 (E, F) in coronal sections of
wildtype (A, C, E) and Nfib knockout (B, D, F) brains. At E14
in the wildtype (A), GLAST was expressed in the glial wedge
(arrow), and this expression intensified as development pro-
ceeded (arrows in (C, E)). Furthermore, GLAST expression
was observed in the indusium griseum glia (arrowhead) and
midline zipper glia (double arrowhead) at E18 in the wildtype
(E). In the mutant, however, GLAST expression in the glial
wedge was reduced in comparison to controls (open arrow-
heads; compare (B) to (A), and (D) to (C)). Moreover, the
indusium griseum glia and midline zipper glia in the Nfib null
mutant were not apparent in the mutant via GLAST immuno-
histochemistry at E18 (F). Scale bars: 300 μm (A, B); 250 μm
(C, D); 200 μm (E, F).

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ically localised to cingulate cortex axons, Npn1 [8], was
diminished in Nfib-deficient mice in comparison to
wildtype controls (Figure 7C, D). Levels of Npn1 mRNA
were also significantly lower in the cortex of Nfib mutants
at E16, the age at which Npn1-expressing axons from cin-
gulate pioneer neurons initiate CC formation (P < 0.05, t-
test; Figure 7J).
We have recently postulated that Npn1-semaphorin sig-
nalling may be vital for callosal formation [8]. 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 cor-
tex contralateral to the injection site were both NFIB- and
True Blue-positive (Figure 7G-I), indicating that a propor-
tion 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 [35]. Analysis of this
pathway with immunohistochemistry against the axonal
marker neurofilament revealed no defect, as in both
wildtype and Nfib-deficient sections, neurofilament-posi-
tive 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
Nfib-deficient mice have been reported to exhibit callosal
agenesis at E17, as axons expressing the cell adhesion mol-
ecule L1 reach, but do not cross, the midline in the
absence of Nfib [22]. L1 is a non-specific marker for axons,
and labels axons from both the cingulate cortex and neo-
cortex. As our DCC labelling had indicated that cingulate
cortex axons extend towards the cortical midline (Figure
7B), it remained a possibility that the L1-positive axons
previously reported at the midline [22] arose not from the
neocortex, but from the cingulate cortex. To investigate
the origin of axons arriving at the midline, we performed
tract tracing by injecting the carbocyanine dye DiI into the
neocortex of both wildtype and Nfib-deficient mice at
E18. Coronal sections at rostral and caudal levels in the
wild type indicated that DiI-labelled axons were coursing
through the CC and ascending into the contralateral hem-
isphere (Figure 8A, B, E, F). At rostral levels in the mutant,
DiI-positive axons could be seen approaching, but not
crossing, the cortical midline (Figure 8C, D). Further-
more, at E18 most callosally projecting neocortical axons
located in layers II/III and V, identified by the expression
of Satb2 [5], did not express NFIB (Additional file 3).
These data indicate that, in the absence of Nfib, neocorti-
cal callosal axons are able to reach the midline, implying
that callosal malformation in Nfib mutants does not arise
from cell-autonomous defects within neocortical neu-
rons. Unexpectedly, at more caudal levels, we observed a
small population of axons crossing the cortical midline in
the Nfib mutants (Figure 8G, H), indicating that, at this
age, Nfib-deficient mice exhibit dysgenesis, not agenesis,
of the CC. These findings were supported by analysis of
the axonal marker GAP43, which indicated that there is
indeed a population of axons that cross the midline via
the CC at caudal levels in mice lacking Nfib (Figure 8I-P).
Reduced expression of Slit2 at the cortical midline of Nfib
mutants
Mice lacking the guidance molecule Slit2 exhibit callosal
defects [9], and expression of Slit2 is also reduced within
the glial wedge of Nfia mutants [20]. Given the dysgenic
phenotype of Nfib-deficient mice, we next examined the
expression of Slit2 to determine whether the phenotype
we observed could be related to altered expression of this
molecule. At E18, expression of Slit2 mRNA in the
wildtype was observed within the glial wedge (Figure 9A).
At rostral sections in the mutant, however, Slit2 expres-
sion was markedly lower (Figure 9B), whereas at more
caudal levels it was at a level more comparable to that seen
in controls (Figure 9C), which could contribute to the for-
mation of the CC caudally in the mutant. Next, we ana-
lysed expression of GFAP more caudally in Nfib-deficient
The subcallosal sling fails to form correctly in Nfib knockout mice
Figure 6
The subcallosal sling fails to form correctly in Nfib
knockout mice. (A-D) Coronal sections of E18 brains
showing expression of Emx1 (A, B) and NFIA (C, D). In the
wildtype, cells of the subcallosal sling were seen crossing the
midline immediately ventral to the CC (arrows in (A, C)). In
the mutant, however, cells of the sling did not cross the mid-
line, and instead remained ipsilateral (arrowheads in (B, D)).
Scale bar: 200 μm.

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Figure 7
Expression of guidance receptors on the axons of cingulate pioneering neurons. (A-F) Expression of DCC (A, B),
Npn1 (C, D) and neurofilament (E, F) in coronal sections of E18 wildtype (A, C, E) and Nfib-deficient (B, D, F) brains. Axons
from neurons in the cingulate cortex initiate callosal tract formation, and express the guidance receptor DCC. Expression was
seen on axons from the cingulate cortex (Cing) in both the wildtype and Nfib null mutant (arrows in (A, B)). However, expres-
sion of Npn1, another guidance receptor localised to cingulate pioneering axons, was diminished in the knockout in compari-
son to littermate controls (arrowheads in (C, D)). The perforating pathway (PP), shown via expression of the axonal marker
neurofilament in wildtype sections (arrow in (E)), appeared relatively normal in mice lacking Nfib (arrow in (F)). (G-I) The ret-
rograde tracer True Blue was injected into the cingulate cortex of E17 wildtype embryos in utero. At E18, immunohistochem-
istry on coronal sections against NFIB (G) demonstrated that the retrograde tracer (H) and NFIB were co-localised in a
population of callosally projecting neurons in the cingulate cortex (arrowheads in (I)). (J) At E16, levels of Npn1 mRNA were
significantly lower in Nfib mutants than in littermate controls (*P < 0.05; t-test). RNA from three independent replicates for
both wild type (WT) and Nfib mutants (Nfib knockout (KO)) was quantified. Error bars indicate standard error of the mean.
IZ, intermediate zone. Scale bar: A-F 200 μm; G-I 30 μm.

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mice, to determine whether there were more mature glia
within the region in which the callosal axons cross the
midline. At rostral levels, few GFAP-positive glia were
detected within the glial wedge region (Figure 9E). In
more caudal regions, however, more GFAP-positive fibres
were observed within the glial wedge, and GFAP-express-
ing cells were also found within the indusium griseum
(Figure 9F). Finally, we performed co-immunofluorescent
labelling of E18 Nfib-/- brains with both GAP43 and GFAP.
At rostral levels in the mutant, GFAP-positive glia were
only observed within the glial wedge, and no axons were
seen crossing the midline (Figure 9G-I). More caudally,
however, labelling revealed GFAP-positive glia within
both the glial wedge and indusium griseum, and callosal
axons were observed crossing the midline (Figure 9J-L).
Collectively, these data suggest that callosal formation in
caudal regions of the Nfib mutant may be facilitated by
the development, albeit delayed, of glial populations.
Defects in multiple forebrain commissures in Nfib mutants
How the lack of Nfib affects the development of other
forebrain commissures such as the hippocampal commis-
sure and the anterior commissure is unknown. To address
forebrain commissure development in Nfib-deficient
mice, we performed diffusion tensor magnetic resonance
imaging (DTMRI) to visualise the major axon tracts
within the brain [36]. The axon tracts identified by DTMRI
were colour-coded to denote the direction in which the
fibre bundles project (blue, dorso-ventrally; red, medio-
laterally; green, rostro-caudally). In a sagittal view of the
midline of an E18 wildtype brain, all three major fore-
brain commissures (CC, hippocampal commissure and
anterior commissure) were evident (Figure 10A). In the
mutant, however, the anterior commissure was absent,
and the CC and the hippocampal commissure, although
evident, were reduced in comparison to that of the control
(Figure 10B). This result was confirmed using tractogra-
phy, which demonstrated a marked reduction in axon
bundles within the hippocampal commissure of Nfib-
deficient mice (Figure 10C, D). Furthermore, conven-
tional analysis of these scanned brains using immunohis-
tochemistry against GAP43 indicated the absence of the
anterior commissure in the mutant (Figure 10E-H).
Discussion
The Nfi gene family is required for multiple aspects of cen-
tral nervous system development [37]. Here we show that
Nfib regulates the differentiation of specific glial popula-
tions 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 cur-
tailed, 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 con-
trols. Collectively, these findings demonstrate that pre-
dominantly 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 play-
ers in glial lineage determination. Nfi genes have previ-
ously been shown to promote the expression of a suite of
glial-specific genes in vitro, including those encoding
GFAP [38], brain lipid binding protein [39], α1-antichy-
motrypsin [40] and tenascin C [41]. 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 [21], while the onset
of gliogenesis in the chick spinal cord requires the action
of Nfia [19]. A mechanistic insight into how Nfi genes
could promote gliogenesis within cortical precursors has
recently been proposed [42]. Namihira and colleagues
[42] 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 down-
regulated 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 high-
lighted significant roles for Nfi family members in neuro-
nal development. For instance, Nfib has been shown to
play a central role in precerebellar mossy fibre neuron
generation within the pons [26], while Nfia controls axon
outgrowth, dendritogenesis and migration of cerebellar

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granule neurons via regulation of N-cadherin and ephrin
B1 [44].
Our finding that a rudimentary CC forms caudally in Nfib-
deficient mice appears inconsistent with the initial charac-
terisation of this knockout line, which reported agenesis
of the CC [22]. 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 previ-
ous 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 [22]. 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-spe-
cific manner, thereby enabling further investigation of CC
formation postnatally in the absence of this transcription
Nfib-deficient mice exhibit callosal dysgenesis
Figure 8
Nfib-deficient mice exhibit callosal dysgenesis. (A-H) Carbocyanine tract tracing in wildtype (A, B, E, F) and Nfib mutant
(C, D, G, H) brains at E18. DiI was injected into the neocortex of wildtype and knockout brains, thereby labelling all neocorti-
cal projections, including the CC. In the wildtype at rostral (A, B) and caudal (E, F) levels, callosal axons were seen projecting
across the midline (arrows in (B, F)). In the knockout at rostral levels, no axons were observed crossing the midline (arrow-
head in (D)). However, at more caudal levels, a small number of axons were seen crossing into the contralateral cortex (arrow
in (H)). (I-P) Immunohistochemistry against the axonal marker GAP43 in wildtype (I, J, M, N) and Nfib mutant (K, L, O, P)
brains at E18. The CC was clearly observed in the wildtype at rostral and caudal levels (arrows in (J, N)). In the mutant at ros-
tral levels, no GAP43-positive axons were seen crossing the midline. Instead, axons stopped adjacent to the midline (arrow-
heads in (L)). More caudally, however, a small CC was evident in the mutant (arrow in (P)). Panels (B, D, F, H, J, L, N, P) are
higher magnifications of the boxed regions in (A, C, E, G, I, K, M, O), respectively. Scale bars: 500 μm (A, C, E, G, I, K, M, O);
200 μm (B, D, F, H, J, L, N, P).

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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 commis-
sure as a substrate to cross the cortical midline [48].
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 devel-
opment 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 indu-
sium griseum at late gestation [23], and furthermore, Nfia
and Nfib are expressed in the same cells within the glial
wedge at E18 (data not shown). Thus, although differen-
tiation 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-autono-
Slit2 expression at the cortical midline
Figure 9
Slit2 expression at the cortical midline. (A) In the wildtype at E18, expression of the axon guidance cue Slit2 was
observed within the glial wedge region (arrows in (A)). (B) In the Nfib null mutant at rostral levels, expression of Slit2 was
diminished (arrowhead). (C) At more caudal levels in the mutant, expression of Slit2 in the glial wedge region was more
noticeable (arrows). (D) Expression of GFAP in the wildtype at E18. (E) In Nfib knockout sections at rostral levels, GFAP was
observed in the glial wedge (arrows). (F) Further caudally, GFAP was observed in both the glial wedge (arrows) and indusium
griseum glia (open arrowhead). (G-L) Co-immunofluorescent labelling of Nfib knockout sections at rostral (G-I) and caudal (J-
L) levels with the axonal marker GAP43 (green) and the astrocytic marker GFAP (red). At rostral levels, no callosal axons
could be seen crossing the midline, and few GFAP-positive glia were observed within the glial wedge (arrowheads in (H, I)). At
caudal levels, however, more GFAP-positive glia were detected within the glial wedge (arrowheads in (K, L)) and GFAP-posi-
tive glia were also seen within the indusium griseum (double arrowheads in (K, L)). Callosal axons are also seen crossing the
midline at this level (arrows in (J, L)). Scale bar: 200 μm.

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Figure 10
Hippocampal commissure formation in Nfib null mutants. (A-D) Colour-coded anisotropy maps of E18 wildtype (A,
C) and Nfib-deficient (B, D) brains. The colour code indicates the direction of axon fibre tracts (blue, dorso-ventrally project-
ing tracts; red, medio-laterally projecting tracts; green, rostro-caudally projecting tracts). Sections in (A, B) are mid-sagittal
views. In the wildtype (A), the three major forebrain commissures were evident; the corpus callosum (CC), the hippocampal
commissure (HC) and the anterior commissure (AC). In the Nfib mutant, the anterior commissure was absent, but the corpus
callosum and the hippocampal commissure were evident, although much reduced in size. (C, D) Coronal views of the brains
scanned in (A, B), in which tractography (yellow lines) was performed on the hippocampal fimbria. The tracts to the hippocam-
pal commissure and the fornix (FX) could be seen at this rostro-caudal position. In the Nfib-deficient brain (D), the size of the
hippocampal commissure was reduced in comparison to that of the wildtype control (C). (E-H) The brains represented in (A,
B) were cut coronally and axon tracts were revealed via expression of the axonal marker GAP43. In the wildtype (E, G), the
hippocampal commissure and anterior commissure were seen crossing the midline. In the Nfib mutant (F, H), a reduced hip-
pocampal commissure was revealed by GAP43 immunohistochemistry, and the anterior commissure was absent. Panels (G, H)
are higher magnifications of the boxed regions in (E, F), respectively. Scale bars: 800 μm (A, B); 500 μm (C-F); 200 μm (G, H).

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mous mechanisms, such as the development of midline
glial and neuronal sling populations, may underlie the
formation of all telencephalic commissures. In conclu-
sion, our data provide a comprehensive insight into the
phenotypic abnormalities underlying callosal malforma-
tion in Nfib-deficient mice, and demonstrate that multiple
factors contribute to these defects during embryogenesis.
Materials and methods
Animals
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 [22] 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 over-
night. The following day was designated as E0 if the
female had a vaginal plug. Embryos were genotyped by
PCR as previously described [22].
Fixation
On the required gestational day, embryos were drop-fixed
in 4% paraformaldehyde (PFA; E14 and below) or tran-
scardially perfused with 0.9% phosphate buffered saline,
followed by 4% PFA (E15 to E18). They were then post-
fixed in 4% PFA at 4°C until sectioning.
Haematoxylin staining
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 haematoxy-
lin using standard protocols.
Immunohistochemistry on floating sections
Brains were sectioned as described above, then processed
free-floating for immunohistochemistry using the chro-
mogen 3,3' diaminobenzidine as described previously
[49]. Primary antibodies used for immunohistochemistry
were anti-GAP43 (mouse monoclonal, 1/100,000;
Chemicon, Bedford, MA, USA), anti-GFAP (rabbit poly-
clonal, 1/50,000; DAKO, Glostrup, Denmark), anti-
cleaved caspase 3 (rabbit polyclonal, 1/1,000; Cell Signal-
ing Technology, Danvers, MA, USA), anti-GLAST (rabbit
polyclonal, 1/50,000; a gift from Niels Danbolt, Univer-
sity of Oslo), anti-nestin (mouse monoclonal, 1/1,500;
Developmental Studies Hybridoma Bank), anti-tenascin
C (rabbit polyclonal, 1/2,000; Chemicon), anti-Tbr1 (rab-
bit polyclonal, 1/100,000; a gift from Robert Hevner, Uni-
versity of Washington), anti-NFIA (rabbit polyclonal, 1/
30,000; Active Motif, Carlsbad, CA, USA), anti-Emx1 (rab-
bit 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 Hop-
kins 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, Burlin-
game, 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 embed-
ded 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' diaminoben-
zidine 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 mid-
line, sections from E13, E14 and E15 wildtype C57Bl/6J or
Nfib-/- embryos were labelled with an anti-phosphohis-
tone 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-posi-
tive cells per unit area at the cortical midline, a 300 μm2-
boxed region, encompassing the presumptive glial wedge

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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).
Statistical analysis
For all experiments described in this study, sections from
three different brains of each genotype were analysed. Sta-
tistical 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% solu-
tion 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- imag-
ing 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 Sci-
entific, 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 previ-
ously [50], with minor modifications. An antisense ribo-
probe 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 tran-
scribed with random hexamers. qPCR reactions were car-
ried 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 fol-
lowed 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 Sci-
ence) 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 trip-
licate. 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-dimen-
sional 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 com-
posed of two Bo and thirty direction diffusion-weighted
images (b value of 5,000 s/mm2, δ/Δ = 2.5/14 ms). Recon-
struction and tractography were performed with Diffusion
Toolkit [51] according to high angular resolution diffu-
sion (HARDI) and Q-ball models [52]. Tractography lim-
its 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 [53].
Abbreviations
CC: corpus callosum; DCC: Deleted in colorectal cancer;
DTMRI: diffusion tensor magnetic resonance imaging; E:
embryonic day; GFAP: glial fibrillary acidic protein;
GLAST: astrocyte-specific glutamate transporter; Npn1:

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neuropilin 1; PFA: paraformaldehyde; qPCR: quantitative
real-time PCR.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MP and LJR conceived the study, evaluated the findings,
prepared the figures and the manuscript. RMG generated
the Nfib mutant strain. MP, RXM, CL, EL, GB, SM, NS and
NDK carried out all the experimental procedures.
Additional material
Acknowledgements
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 sup-
ported 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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Additional file 1
Figure S1 - Co-expression of NFIB and β-galactosidase in Nfib heter-
ozygous mice. (A-I) Confocal image of an E18 Nfib heterozygote brain
demonstrating expression of (β-galactosidase (β-gal, A, D, G) and NFIB
(B, E, H) at the cortical midline. (A-C) Low power image of the cortical
midline, demonstrating extensive co-expression of β-gal and NFIB (C).
(D-F) Higher magnification view of the glial wedge (GW), demonstrat-
ing that the majority of cells in this region co-express NFIB and β-gal, in
particular those cells located at the ventricular surface (arrowhead in F).
(G-I) Higher magnification view of the indusium griseum glia (IGG),
showing that these cells express both NFIB and β-gal (arrows in I). Scale
bar: A-C 200 μm; D-I 100 μm.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1749-
8104-4-43-S1.TIFF]
Additional file 2
Figure S2 - Reduction of tenascin C expression in the absence of Nfib.
Coronal sections of wildtype (A, C) and Nfib-deficient (B, D) brains.
Expression of the glial marker tenascin C was reduced at the cortical mid-
line of mice lacking Nfib at both E15 (B) and E16 (D). Scale bar: A, B
280 μm; C, D 250 μm.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1749-
8104-4-43-S2.TIFF]
Additional file 3
Figure S3 - Expression of NFIB and Satb2 in the neocortex. Confocal
image of an E18 wildtype brain. (A) DAPI nuclear staining; the boxed
region indicates the position of panels B-D. Satb2, a marker for callosally
projecting axons, was expressed predominantly in upper layers of the cor-
tical plate (B). NFIB expression was highest in the deeper cortical layers,
but was also seen in layers II/III (C). Most cells within the cortical plate
did not co-express NFIB and Satb2 (D), although some overlap was evi-
dent in layers II/III. Scale bar: A 500 μm; B-D 100 μm.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1749-
8104-4-43-S3.TIFF]