Morphological and functional midbrain phenotypes in Fibroblast Growth Factor 17
mutant mice detected by Mn-enhanced MRI
Xin Yua,1, Brian J. Niemana,2, Anamaria Sudarove, Kamila U. Szulca, Davood J. Abdollahiana, Nitin Bhatiad,
Anil K. Lalwanid, Alexandra L. Joynere, Daniel H. Turnbulla,b,c,⁎
aKimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York NY, USA
bDepartments of Radiology, New York University School of Medicine, New York NY, USA
cDepartment of Pathology, New York University School of Medicine, New York NY, USA
dDepartment of Otolaryngology, New York University School of Medicine, New York NY, USA
eDevelopmental Biology Program, Sloan-Kettering Institute, New York NY, USA
a b s t r a c ta r t i c l ei n f o
Received 22 November 2010
Revised 14 February 2011
Accepted 17 February 2011
Available online 26 February 2011
With increasing efforts to develop and utilize mouse models of a variety of neuro-developmental diseases,
there is an urgent need for sensitive neuroimaging methods that enable in vivo analysis of subtle alterations in
brain anatomy and function in mice. Previous studies have shown that the brains of Fibroblast Growth Factor
17 null mutants (Fgf17−/−) have anatomical abnormalities in the inferior colliculus (IC)—the auditory
midbrain—and minor foliation defects in the cerebellum. In addition, changes in the expression domains of
several cortical patterning genes were detected, without overt changes in forebrain morphology. Recently, it
has also been reported that Fgf17−/−mutants have abnormal vocalization and social behaviors, phenotypes
that could reflect molecular changes in the cortex and/or altered auditory processing / perception in these
mice. We used manganese (Mn)-enhanced magnetic resonance imaging (MEMRI) to analyze the anatomical
phenotype of Fgf17−/−mutants in more detail than achieved previously, detecting changes in IC, cerebellum,
olfactory bulb, hypothalamus and frontal cortex. We also used MEMRI to characterize sound-evoked activity
patterns, demonstrating a significant reduction of the active IC volume in Fgf17−/−mice. Furthermore, tone-
specific (16- and 40-kHz) activity patterns in the IC of Fgf17−/−mice were observed to be largely overlapping,
in contrast to the normal pattern, separated along the dorsal-ventral axis. These results demonstrate that
Fgf17 plays important roles in both the anatomical and functional development of the auditory midbrain, and
show the utility of MEMRI for in vivo analyses of mutant mice with subtle brain defects.
© 2011 Elsevier Inc. All rights reserved.
Several secreted proteins in the Fibroblast Growth Factor (Fgf)
family are critical for neural patterning during embryogenesis. Among
these, Fgf8 has been shown to be the main molecule responsible for
“organizer” activity–the ability of a cell or tissue to send signals that
instruct the fates of surrounding cells—during development of the
mid-hindbrain (MHB) region (Crossley et al., 1996; Lee et al., 1997;
Ye et al., 1998; Sato et al., 2001), and is also critical for normal
forebrain development (Shimamura and Rubenstein, 1997; Meyers
et al., 1998; Ye et al., 1998; Fukuchi-Shimogori and Grove, 2001; Garel
et al., 2003; Storm et al., 2006). Fgf17 expression overlaps with Fgf8,
and also plays a role in the development of the MHB and cortex
(Hoshikawa et al., 1998; Maruoka et al., 1998; Xu et al., 1999, 2000;
Reifers et al., 2000; Liu et al., 2003; Fukuchi-Shimogori and Grove,
2003). While Fgf8−/−mice are embryonic lethal, Fgf17−/−mice are
viable and fertile, having mild defects in the MHB, including (non-
quantified) reduction of the inferior colliculus (IC), and small foliation
changes in the cerebellum (Xu et al., 2000). Fgf17−/−mutants also
show altered expression domains of genes that mark sub-regions of
frontal cortex, without any obvious change in forebrain morphology
(Cholfin and Rubenstein, 2007, 2008).
Interestingly, Fgf17−/−mice exhibit abnormal social behaviors,
including decreased vocalization in pups and deficits in several adult
behaviors involving social interactions, reminiscent of autism in
humans (Scearce-Levie et al., 2008). It was suggested that these
abnormal behaviors may result from altered forebrain function, based
NeuroImage 56 (2011) 1251–1258
Abbreviations: ABR, auditory brainstem recording; DBM, deformation based
morphometry; Fgf17, fibroblast growth factor 17; IC, inferior colliculus; MRI, magnetic
resonance imaging; MEMRI, manganese (Mn)-enhanced MRI.
⁎ Corresponding author at: Skirball Institute of Biomolecular Medicine, New York
University School of Medicine, 540 First Avenue, New York, NY 10016, USA. Fax: +1
212 263 8214.
E-mail address: Daniel.Turnbull@med.nyu.edu (D.H. Turnbull).
1Current address: Laboratory of Functional and Molecular Imaging, National
Institute of Neurological Disorders and Stroke, Bethesda, MD, USA.
2Current address: Mouse Imaging Centre, Hospital for Sick Children and Toronto
Centre for Phenogenomics, Toronto ON, Canada.
1053-8119/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
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on evidence of reduced activation of the immediate early-gene Fos in
Fgf17−/−frontal cortex after exploration of novel environments
(Scearce-Levie et al., 2008). However, it is unclear whether altered
auditory function, as a result of the known defects in the IC, the
auditory midbrain, also contributes to the behavioral phenotypes in
We hypothesized that whole brain anatomical MRI analysis would
provide a more complete and quantitative characterization of the
Fgf17−/−mouse morphological phenotype than previous qualitative
studies based on histology. We also sought to investigate the
possibility that Fgf17−/−mice have altered auditory function as a
result of the reduction in IC. Previously, we have shown that MEMRI
can be used to detect and analyze sound-evoked activity patterns in
the mouse IC (Yu et al., 2005, 2007, 2008), based on the activity-
dependent accumulation of paramagnetic Mn ions in neural cells via
uptake through voltage-gated calcium channels (Lin and Koretsky,
1997). In addition to characterizing the normal mouse IC, we also
demonstrated the utility of MEMRI to analyze altered IC activity
patterns in mice exposed to atypical sound-rearing environments
during auditory brain development (Yu et al., 2007). In the current
study, we employed similar MEMRI-based functional analysis
approaches to characterize differences in IC activity patterns,
comparing Fgf17−/−mice and control littermates. Our findings of
altered auditory function in Fgf17−/−mutants may help to explain at
least some of the behavioral phenotypes that have been reported in
Materials and methods
All mice used in these studies were maintained under protocols
approved by the Institutional Animal Care and Use Committee of New
York University School of Medicine. Fgf17 mutant mice were obtained
from the Ornitz lab (Washington University), and Fgf17+/−x Fgf17+/−
breeding pairs were mated to produce littermate Fgf17+/+wildtype
(WT), Fgf17+/−heterozygous and Fgf17−/−homozygous mutants,
while Fgf17−/−×Fgf17+/−breeding pairs were mated to produce
littermate Fgf17+/−and Fgf17−/−mice. Genotype analysis was
performed via polymerase chain reaction from tail DNA, using primers
detect the mutant allele, and r1:GACAGCAGAGAATCAATAGCTGC; r2:
GAAGTTTCTCCAGCGATGGG to detect wild type Fgf17, as described
previously (Basson et al., 2008).
For MEMRI, WT, Fgf17+/−, and Fgf17−/−mice were injected
intraperitoneally (IP) with an aqueous solution of MnCl2(0.4 mmol/
kg) one day before imaging (Yu et al., 2005, 2007). After injection, mice
were placed in a controlled acoustic environment inside an acoustic
isolation chamber (Mac-1, Industrial Acoustics) for 24-h. During this
12-h light / 12-h dark cycle and free access to food and water. Neither
MnCl2administration, nor sound stimulation, induced any obvious
abnormal behavior during the 24-h time period.
Auditory brainstem recordings
Auditory brainstem recordings (ABR) were measured using calibrat-
ed equipment and protocols, as described in detail by Willott (2006).
Prior to ABR testing, mice were anesthetized with an intramuscular
injection of a mixture of ketamine (50 mg/kg) and xylazine (95 mg/kg).
ABR testing was performed in a double-walled acoustic chamber
(Industrial Acoustics), using an isothermic heating pad (K20, Baxter) to
maintain body temperature at 36–38 °C. Silver electrodes were inserted
subcutaneously at the vertex of the skull, and just inferior to each ear
(ispilateral=reference; contralateral=ground). The biological signal,
between the vertex and reference electrodes, was amplified (DAM-50E,
WorldPrecisionInstruments),bandpassfilteredfrom0.3to3 kHz,90 dB/
octave, (VBF8.04, Stewart Electronics), digitized at a 25-kHz sampling
rate and recorded over a 10-ms window (System II, Tucker Davis
Technologies). Response was measured to both click and pure tone (8-,
16-, 24- and 32-kHz) stimuli, decreasing the amplitude in 5-dB steps,
from 95 to 5 dB peak sound pressure level (SPL). Threshold was
determined as the level one step above that where no discernible ABR
waveform was detected. Differences in the threshold data for the three
genotypes were analyzed statistically with ANOVA (one-way). Note that
32-kHZ was the highest frequency that could be achieved reliably with
the existing ABR recording system.
MRI data acquisition
et al., 2005, 2007). Briefly, T1-weighted brain images were acquired
with a 3D gradient echo pulse sequence (echo time, TE=4-ms;
repetition time, TR=50-ms; flip angle=65°), resulting in a volumetric
image set covering the entire brain, with isotropic spatial resolution of
100-μm in a total imaging time of 110 min per mouse. MRI was
performed on a micro-imaging system (SMIS) consisting of a 7-Tesla
horizontal magnet (Magnex Scientific) with actively shielded gradients
(Magnex: 250-mT/m gradient strength; 200-μs rise time) and a 25-mm
(inner diameter) quadrature Litzcage mouse headcoil (DotyScientific).
where P0 denotes the day of birth.
Acoustic stimulation protocol
The acoustic environment and sound stimuli were described in
detail previously (Yu et al., 2005, 2007). Sound stimulation in the
current study consisted of calibrated broadband sound (1–59 kHz)
and single tones (16- and 40-kHz) played through a speaker mounted
inside the acoustic isolation chamber. The carrier frequencies were
amplitude modulated at 4-Hz with a modulation range of 90%. For all
experiments, the amplitude-modulated stimuli had peak amplitudes
of 89 dB SPL, measured at the calibration point in the geometrical
center of the cage, 10-cm above the cage bottom (Yu et al., 2007).
injection of MnCl2(0.4-mmol/kg, IP) at P20 and 24 h of exposure to 40-
kHz. The same mice were subsequently kept in a quiet environment for
mmol/kg, IP) at P23 for imaging at P24after 24 h of exposure to 16-kHz.
For comparison, a separate group of Fgf17−/−mice was imaged at P21,
16-kHz activity patterns at age P21 and P24 were compared to
determine the effects of mouse age and the longitudinal experimental
protocol on the MEMRI enhancement patterns and the resulting
Histology and immunohistochemistry
Mice were anesthetized via IP injection of a mixture of ketamine
(100-mg/kg) and xylazine (10-mg/kg) in phosphate buffered saline
(PBS) and perfused transcardially with PBS, followed by 4% parafor-
maldehyde. For frozen sections, the brains were removed quickly,
post-fixed for 24-h in 4% paraformaldehyde at 4 °C, and cryoprotected
in (4 °C) 15% and 30% sucrose solution, successively. After embedding
in OCT media (Tissue-Tek, Sakura), frozen 12-μm sections were cut on
a cryostat for histological analysis. Immunohistochemistry was
performed using standard staining procedures with Neurogranin
primary antibody (1:1000; Chemicon), followed by an Alexa-555-
conjugated donkey anti-rabbit secondary antibody (Molecular
Probes). For cell density analysis, the fixed brains were embedded
in paraffin and 20-μm sections were cut on a microtome. To assay the
number of cells in the IC, we quantified 3 cresyl violet stained sections
X. Yu et al. / NeuroImage 56 (2011) 1251–1258
of the medial IC in each mouse using NIH ImageJ software, counting
cells in an area of 1.65-mm2located centrally along the dorsal-ventral
axis. Cells were chosen for quantification only if the nucleus was fully
visible and in focus.
Analysis of whole brain and IC volume
As an initial comparison of anatomical phenotypes, we performed
volumetric analyses of segmented brain and IC volumes. Using the
propagating active contour filter in Amira software (TGS), whole brains
were segmentedsemi-automatically, usingmanualeditingtorefinethe
contours from each individual Fgf17+/−and Fgf17−/−mouse. Whole
brain templates were created using rigid, 12-parameter affine
transformation for registration and averaging the groups of Fgf17−/−
and Fgf17+/−mice separately. The registered mouse brain images were
then normalized, adjusting the whole brain histograms to obtain equal
mean and standard deviation (for later intensity-based analyses). ICs
were extracted from the processed brain images using 3D IC masks
created separately for Fgf17−/−and Fgf17+/−mice. The dorsal-caudal
borders of the IC were easily identified by the adjacent cerebral spinal
fluid (CSF) and cerebellum. The rostral border was identified by the
decrease in Mn-enhancement in the IC compared to the superior
colliculus, while the ventral borders were defined by the nucleus of the
branchium of the IC and the CSF in the midbrain aqueduct. Using the
resulting average masks, the IC was segmented, and the volume
computed as the product of the voxel volume with the number of IC
voxels, for each mouse. Whole brain and IC volumes were computed as
respectively. Differences in volume were analyzed statistically using
Student's t-test (two-way), with significance set at pb0.05.
Fgf17+/−and Fgf17−/−mice were analyzed for anatomical differ-
ences with unbiased deformation-based morphometry (DBM), using
software obtained from the Mouse Imaging Centre (Toronto, Canada),
the Montreal Neurological Institute (Collins et al., 1994). At completion
of the registration process, local volume differences were identified
computationally without prospective identification of anatomical
structures. DBM methods for anatomical phenotyping in mice have
been described previously (Kovacevic et al., 2005; Nieman et al., 2006,
2007; Lerch et al., 2008). The generation of a reference average image
began by normalization of the orientation, location and intensity of all
component images. Subsequently, an average space was defined that
pair-wise image registrations (Woods et al., 1998). After generating an
at each voxel, each image was re-registered totheaverage iteratively to
generate progressively refined averages. At each iteration, the registra-
tion was performed at a finer scale, until the final average with full-
resolution (100-μm) was achieved. The final set of deformations from
the Fgf17+/−and the Fgf17−/−groups to the average were then
analyzed for significant anatomical differences. For volumetric differ-
ences, the local size changes encoded by the deformations were
scale factor that represents voxel volume changes. A voxelwise
Student's t-test of log-Jacobian values was used to identify regions of
statistically significant change, correcting for multiple statistical
comparisons using the false discovery rate (Benjamini and Hochberg,
Analysis of IC activity
The image analysis protocols for analyzing IC activity have been
described in detail previously (Yu et al., 2008). Statistical analysis of
MEMRI signals in the IC was performed voxel-wise with a two-tailed
Student's t-test to compare the active IC volume between broadband
sound stimulated and quiet control mice (Yu et al., 2005, 2008). To
study the tonotopic organization within the IC, we analyzed the active
IC volume after exposing mice to pure 16- and 40-kHz tones. In these
experiments, maps of signal hyperintensity were created by setting a
signal intensity (SI) threshold to be SIMean+1.5 SISD, where the mean
(SIMean) and standard deviation (SISD) were measured from the
histogram for each IC volume. The location of the active IC under pure
toneconditionswaslocated withinthe wholeIC volumeby measuring
the distance between the centroids of each volume. Comparisons of
the locations of the 16- and 40-kHz activity patterns in Fgf17−/−and
Fgf17+/−mice were then made on the basis of their centroid locations
(after averaging the left and right centroid locations for each mouse
individually). For visualization of the variability between mice, the
standard deviations of the centroid positions on each principal axis
were used to define the radii of an ellipsoid, and the ellipsoids for the
16- and 40-kHz activity patterns were overlaid to show both the
location and variability of the centroids for each activity pattern.
Differences in centroid separation were analyzed statistically using
Student's t-test (two-way), with significance set at pb0.05.
Region-of-interest (ROI) measurements were also made in the
cochlear nucleus, as described previously (Yu et al., 2005), to
determine whether there were any differences in the auditory input
circuit, upstream of the IC, comparing Fgf17−/−and Fgf17+/−mice.
ABR recordings show no peripheral hearing defects in Fgf17 mutant mice
ABR thresholds were measured in 4-week old WT(N=4), Fgf17+/−
(N=4) and Fgf17−/−(N=4) littermates (Fig. 1). The 4-week age was
chosen as the earliest developmental stage that consistent ABR
recordings could be acquired with the existing experimental setup.
or pure tone (Fig. 1B) stimulation. We concluded that loss of Fgf17 does
not result in hearing impairment in these mice.
Analysis of anatomical brain phenotypes in Fgf17−/−mice
Analysis of whole brain and IC volumes was first performed using
MEMRI images of small groups of P21 WT (N=4) and Fgf17+/−(N=4),
confirming previous reports that there is no obvious phenotype in
heterozygous Fgf17 mutants (Xu et al., 2000). Volumetric and statistical
analyses were then performed in larger groups of Fgf17+/−(N=14) and
Fgf17−/−(N=14) mice, showing no differences in the whole brain
volumes between WT, Fgf17+/−and Fgf17−/−mice (Fig. 2A), or in IC
mice was 17±5% (mean±standard deviation) smaller than in Fgf17+/−
mice (p=0.0001; Fig. 2B), confirming the previous (qualitative)
histological results (Xu et al., 2000). These results provide the first
quantification of the anatomical IC phenotype of Fgf17 knockout mice.
After MEMRI, brains were sectioned for histological analysis,
stainingforNeurogranin, aproteinkinaseC (PKC)substrateexpressed
by IC neurons (Huang et al., 1993; Neuner-Jehle et al., 1996). There
was good qualitative agreement between in vivo MEMRI images
(Figs. 2C,D) and Neurogranin staining (Figs. 2E,F). Note that
Neurogranin is more highly expressed in the IC compared to adjacent
brain regions, allowing identification of the IC nuclei in histological
sections (Figs. 2E,F). Quantitative analysis of IC cell density revealed a
17% increase in Fgf17−/−(N=3) compared to Fgf17+/−(N=3) mice
(Fig. 2G), closely matching the magnitude of the morphological
decrease, and showing that the number of IC cells was not affected by
loss of Fgf17.
To further explore the anatomical phenotypes in Fgf17 mutant
mice, automated and unbiased DBM analysis was performed to
X. Yu et al. / NeuroImage 56 (2011) 1251–1258
compare P21 Fgf17−/−(N=10) and Fgf17+/−(N=15) control mice
(Fig. 3, false discovery rate, FDR=0.05; Table 1). The resulting
deformation maps showed small but statistically significant regional
size differences between the two groups of mice. These included a
reduced volume in the IC and anterior cerebellum in Fgf17−/−mice,
and an increased volume in the fluid space above the IC, consistent
with the IC deletion. In the forebrain, DBM analysis detected an
expansion of the anterior-medial rhinal fissure, extending into the
more lateral cortical regions suggesting possible alterations in the
structure of the frontal cortex, previously undetected by histological
analysis. Finally, the DBM maps showed a significant increase in the
volume of a medial subregion of hypothalamus, and a decrease in the
ventral-caudal olfactory bulb (OB), both previously undetected
phenotypes in Fgf17−/−mutants.
MEMRI reveals a significant decrease in the active IC of Fgf17−/−mice
To assess the functional consequences resulting from loss of Fgf17
during midbrain development, we analyzed regions of enhanced
MEMRI signal in Fgf17+/−and Fgf17−/−mice after periods of defined
sound stimulation. This approach was previously demonstrated to
allow analysis of the functional core of the IC (Yu et al., 2005). For this
analysis, we compared IC signal intensities between control mice kept
in a quiet environment (Fig. 4A; N=7 for each genotype) and mice
stimulated with broadband sound, covering the entire audible
frequency range for mice up to 59-kHz (Fig. 4B; N=7 for each
genotype). Consistent with the ABR data, analysis of signal intensities
in the cochlear nucleus showed no differences between Fgf17+/−and
Fgf17−/−mice (data not shown), indicating that any differences
detected in the IC were not simply due to a global reduced activity in
the auditory inputs of Fgf17−/−mice. Active IC regions for each
genotype were characterized by 3D p-maps (Yu et al., 2008),
consisting of voxels with significantly increased signal intensity
compared to quiet controls (pb0.05, Fig. 4C). There was a 36%
reduction in the average volume of the active IC, comparing Fgf17−/−
(0.38-mm3) and Fgf17+/−(0.60-mm3) mice. When expressed as a
ratio of active-to-total IC volume, the ratio in Fgf17−/−mice was still
more than 20% lower than in Fgf17+/−littermates (Table 2). These
results show that loss of Fgf17 leads to a significant reduction in the
volume of the IC region activated by auditory input, beyond that
predicted by the morphological deletion.
MEMRI reveals a significantly altered IC tonotopic map in Fgf17−/−mice
To further examine the functional organization of the IC in Fgf17
mutants, we compared tone-specific activity patterns in Fgf17+/−and
Fgf17−/−mice. Frequency selectivity is a hallmark feature of the
central auditory system, mainly attributed to the tonotopic projection
of afferents into the IC and other auditory nuclei, which results in
spatial patterning of tone-specific activity in each nucleus. In the
normal IC, the tonotopic map is relatively simple, with lower
frequencies activating more dorsal neurons, while higher frequencies
activate more ventral neurons (Romand and Ehret, 1990). Longitu-
dinal MEMRI experiments were performed to compare the positions
Fig. 1. ABR recordings detected no hearing deficits in Fgf17 mutant mice. ABR threshold
measurements after click stimulation were similar between Fgf17+/+wild type (WT),
Fgf17+/−, and Fgf17−/−mice (N=4 for each genotype), with no significant difference
between genotypes (A). During exposure to different pure tones, ABR threshold
measurements were also similar among the three groups, with no statistically significant
Fig. 2. MEMRI and histology confirmed the reduction of IC in Fgf17−/−mice. 3D MEMRI images of the brain and IC allowed quantitative volumetric analysis. Quantitative analysis
showed no difference between the whole brain volumes of WT (N=4), Fgf17+/−(N=14) and Fgf17−/−mice (N=14) (A), or between the IC volumes of WT and Fgf17+/−mice,
while the IC volume in Fgf17−/−was significantly reduced compared to Fgf17+/−mice (B; *p=0.0001). This reduction in IC size was also evident in sagittal MEMRI images (C,D) and
matched histological (E,F), using Neurogranin (red) staining to identify the IC nuclei (A,B). Nuclear DAPI (blue) staining was used as a counterstain, highlighting the adjacent
cerebellum (Cb). The red inset boxes (C,D) show the approximate position of the histological sections (E,F) relative to the MEMRI images. Quantitative analysis showed a significant
increase in the number of cells per unit area in 3 slices of the central IC, comparing Fgf17+/−(N=3) and Fgf17−/−(N=3) mice (G; *p=0.026).
X. Yu et al. / NeuroImage 56 (2011) 1251–1258
of 16- and 40-kHz IC activity patterns in both Fgf17+/−(N=7) and
Fgf17−/−(N=10) mice. 40-kHz MEMRI data were first acquired in
each mouse at P21. Mice were then kept in a quiet environment for
two days to allow activity induced Mn accumulation to be cleared
from the IC (Yu et al., 2005), after which 16-kHz MEMRI data were
acquired in each mouse at P24. Tone-specific activity patterns were
determined using thresholds based on the signal intensities in the
MEMRI images (Yu et al., 2005).
In Fgf17+/−mice, 16- and 40-kHz activity patterns appeared
normal (Yu et al., 2007), with well-separated domains aligned
tonotopically along the dorsal-ventral axis of the IC (Fig. 5A). In
contrast, the 16- and 40-kHz patterns were largely overlapping in
Fgf17−/−mice, with only a small dorsal displacement of the 16-kHz
region relative to 40-kHz region (Fig. 5B). Analysis of the distribution
of tone-specific centroids(Figs.5C,D)andstatisticalcomparisonofthe
spatial location of the centroids (Figs. 5E,F) both demonstrated and
quantified the significantly reduced separation between the 16- and
40-kHz activity patterns in Fgf17−/−mice compared to Fgf17+/−
controls. Specifically, the (16-40 kHz) centroid-centroid distance
measured in Fgf17−/−mice was 49% smaller than Fgf17+/−controls.
Interestingly, this centroid–centroid separation in Fgf17+/−mice
(mean±standard deviation=351±110 μm) was virtually identical
to the distance measured in WT mice in a previous study (352±
27 μm; Yu et al., 2007), further confirming the lack of a detectable IC
phenotype in Fgf17+/−heterozygous mutants. Taken together, these
results indicate that in addition to the reduction in the size of the
functional IC, there is also a significant alteration in the tonotopic
organization of the IC in Fgf17−/−mutant mice.
Finally, to determine whether the longitudinal imaging protocol
might affect the second set of 16-kHzmeasurements, a separate group
of Fgf17−/−mice (N=6) was tested once only with 16-kHz
stimulation at P21. There were no significant differences between
these data and 16-kHz data from P24 Fgf17−/−mice after 40-kHz
testing at P21 (Suppl. Fig. 1), showing that the longitudinal protocol
used in these investigations was equivalent to testing individual
groups of mice, between P21 and P24, at each frequency.
This study provides the first in vivo quantitative analysis of
morphological and functional defects in the brains of Fgf17 knockout
mice. In comparisons of 3D MEMRI images of Fgf17+/−and Fgf17−/−
mutants, unbiased whole brain DBM analysis demonstrated significant
morphological differences in the IC and cerebellum, regions previously
reported to be altered based on histology (Xu et al., 2000). In addition,
the olfactory bulbs in Fgf17−/−mice were found to be significantly
smaller, while the fissure anterior to the medial frontal cortex, as well
as more lateral frontal cortical regions were larger. This last result
suggests a possible change in the anatomical structure of the frontal
cortex in Fgf17−/−mice, an interesting finding given the altered gene
expression in that region (Cholfin and Rubenstein, 2007), and the
recent report of subtle cortical white matter abnormalities assessed by
diffusion tensor imaging (Moldrich et al., 2010). In the IC there was
good qualitative agreement between MEMRI and histology, while 3D
MEMRI analysis was used to quantify the reduction in the Fgf17−/−IC
volume. Interestingly, quantitative MEMRI activity mapping showed
an even greater decrease in the active IC volume of Fgf17−/−mice after
broadband sound stimulation. Moreover, the organization of IC
neurons receiving auditory afferent projections is likely altered in
Fgf17−/−mice, since the tone-specific (16- and 40-kHz) activity
patterns were abnormal.
One question we aimed to address with our data was what is the
relationship between the functional and anatomical phenotypes in
Fgf17−/−mice. Some reduction in the volume of the active IC core
might be expected in a smaller IC, without necessarily implying an
altered organization of neurons and/or axonal projections in the IC of
Fgf17−/−mice. Arguing against the possibility that the functional
changes are simply byproducts of the altered morphology are the
Fig. 3. DBM analysis revealed morphological differences in Fgf17−/−mice. Slices through 3D MRI datasets show the average Fgf17+/−and Fgf17−/−images, respectively, in the left
and middle column. In the right column, statistically significant voxel size changes are highlighted (5% false discovery rate, FDR). The color scale indicates regions that were
significantly larger (red/yellow; scale goes up to 2×) or smaller (blue; scale goes down to 0.5×) in the Fgf17−/−mice compared to the Fgf17+/−control littermates. Labels:
cerebellum, Cb; frontal cortex, Fc; hypothalamus, Ht; inferior colliculus, IC; olfactory bulb, OB.
Summary of DBM results comparing Fgf17−/−and Fgf17+/−mice. There were
significant volume decreases (−) in the inferior colliculus (IC), cerebellum (Cb) and
olfactory bulb (OB), and significant volume increases (+) in the cerebral spinal fluid
overlying the IC (IC-CSF), anterior-medial rhinal fissure (AM-RF), and the hypothal-
amus (Ht). DBM mapping was performed with a false discovery rate (FDR) of 0.05.
ICIC-CSFCb AM-RF Ht OB
X. Yu et al. / NeuroImage 56 (2011) 1251–1258
relative magnitudes of the observed phenotypes. First, the decrease
in active IC volume after broadband stimulation (36%) was more
than twice the morphological decrease (17%). Furthermore, the
distance between the centroids of the 16- and 40-kHz patterns was
reduced by 49% in Fgf17−/−mice compared to Fgf17+/−mice, a
reduction well beyond that necessary to accommodate either the IC
or active-IC volume changes. It should be noted that the quantitative
parameters derived from the statistical maps, and their relative
magnitudes, should only be interpreted in comparisons between
genotypes (Fgf17−/−vs. Fgf17+/−), and not as absolute measures of
brain activity, as measured, for example by electrophysiology. As
with any statistical test, our choice of the pb0.05 detection
threshold, although conventional, is somewhat arbitrary. Neverthe-
less, the statistical mapping results strongly suggest that the
tonotopic organization of the IC is abnormal in Fgf17−/−mutants,
leading us to speculate on the underlying cause of these changes.
One possibility is that Fgf17 is involved in patterning the tonotopi-
cally ordered afferent innervations in the IC, which could be tested in
future by performing axon-tracing studies, for example to determine
whether defects exist in connectivity between IC and cochlea in
Fgf17−/−mutants. Interestingly, there was no difference in the total
number of IC cells in Fgf17−/−compared to Fgf17+/−mice, again
suggesting that the functional differences are not a simple
consequence of the morphological alterations.
It is also interesting to consider the current results in light of the
behavioral abnormalities reported in Fgf17−/−mutant mice. The major
behavioral phenotypes were decreased vocalizations of the mutant
pups after isolation from their mothers, and decreased interaction time
between adult Fgf17−/−males and wildtype females, and between
Fgf17−/−male–female pairs after extended time in a novel environ-
these behavioral phenotypes were previously attributed to changes in
cortical gene expression, it is equally likely that they result from altered
patterns of auditory activity implied by our data. Future investigation
of auditory function is therefore important for understanding the
In the embryonic MHB, Fgf8 expression is restricted to the anterior
hindbrain, whereas Fgf17 is more broadly expressed in the hindbrain
as well as the posterior midbrain (anlage of the IC), and persists over a
longerdevelopmental window(Xu,etal.,2000;Liuetal.,2003). Based
on mutant studies, Fgf17 is thought to play a secondary role to Fgf8
during embryogenesis, particularly during MHB development. Genet-
ic studies in mutant zebrafish embryos (no isthmus, noi=Pax2.1;
acerebellar, ace=Fgf8) detected no Fgf17 expression in the MHB,
indicating that Pax2.1 and Fgf8 are crucial upstream components in
the pathway that activates Fgf17 (Lun and Brand, 1998; Reifers et al.,
2000). Similarly, in Fgf8 conditional mutant mice, Fgf17 expression is
lost after Fgf8 is ablated (Chi et al., 2003). The separate roles of Fgf17
andtwo Fgf8 isoforms(Fgf8a and Fgf8b)have alsobeen demonstrated
in the mouse MHB. Fgf8b acts as a potent hindbrain organizer
molecule, able to induce hindbrain genes and repress midbrain genes
(Sato et al., 2001; Liu et al., 2003). In contrast, Fgf17, similar to Fgf8a,
primarily induces midbrain structures (Liu et al., 2003). Since Fgf17
expression is lost in Fgf8 mutants, it cannot compensate for loss of
Fgf8, likely accounting for part of the phenotypic difference between
mice lacking Fgf8 (Sun et al., 1999; Shamim et al., 1999; Chi et al.,
2003) or Fgf17 (Xu et al., 2000; Liu et al., 2003).
Our results show that loss of Fgf17 during embryonic brain
development has a significant effect on postnatal brain function.
Given the interactions between Fgf17 and Fgf8 during embryogenesis
described above, it would be interesting in future to assess allelic
series of Fgf mutants, comparing activity patterns in the brains of
Fgf17, Fgf8 andFgf8/17double mutants, as wellas conditional alleles of
both genes. This would enable detailed analyses of the relationships
between embryonic patterning defects due to misregulation of Fgf
signaling and postnatal brain function. The abnormal tonotopic maps
in the Fgf17−/−mice suggest that Fgf signaling is involved in the
guidance of tonotopically organized afferents innervating the IC.
However, it is still unclear whether Fgf17 directly regulates axonal
targeting, or whether it regulates other guidance molecules indirectly.
Thus, the molecular mechanisms of Fgf signaling in the formation of
tonotopic maps remain to be uncovered. This study demonstrates the
Fig. 4. MEMRI revealed a reduced functional IC in Fgf17−/−mice. Comparison of averaged coronal IC MEMRI images of mice maintained in a quiet environment (A) to mice exposed to
broadband (1–59 kHz)soundstimuli(B) revealedMEMRI enhancement afterstimulation inbothFgf17+/−(left) and Fgf17−/−(right) mice. Voxelwise statisticalanalysisidentifiedthe IC
voxels with significant signal enhancement, displayed in 3D p-maps (C; red contours, p≤0.05), showing a reduction in the “active IC” of Fgf17−/−compared to Fgf17+/−mice.
Analysis of IC volumes and active-to-total IC volume ratios in Fgf17−/−and Fgf17+/−
mice. Results are expressed as mean±standard deviation for each entry.
GenotypeIC volume (mm3) Active to total IC volume ratio (%)
X. Yu et al. / NeuroImage 56 (2011) 1251–1258
importance of neuroimaging approaches like MEMRI and DBM for
identifying and quantifying subtle changes in brain morphology, and
for providing results that reveal functional abnormalities beyond the
anatomical phenotypes. This study is also the first demonstration that
MEMRI approaches can be applied to analyze functional phenotypes
in the central auditory system of mutant mice. Importantly, MEMRI
results can be used to guide future studies of altered circuitry and
behavior in a variety of genetically engineered mice.
Supplementary materials related to this article can be found online
This research was supported in part by grants from the NIH
(R01NS038461 and R01HD050767). We thank David Ornitz
(Washington University) for providing the Fgf17 mutant mice used
in these studies, and Dan Sanes (NYU) for insightful discussions
during the course of this work. We also thank Anand Mhatre (NYU
School of Medicine) for advice on the ABR measurements, and Mark
Henkelman, John Sled and Jason Lerch (Mouse Imaging Centre,
TorontoCanada)for providingthesoftware usedfortheDBManalysis.
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