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fnmol-12-00333 January 11, 2020 Time: 11:37 # 1
ORIGINAL RESEARCH
published: 21 January 2020
doi: 10.3389/fnmol.2019.00333
Edited by:
Yi-Ping Hsueh,
Institute of Molecular Biology,
Academia Sinica, Taiwan
Reviewed by:
Shen-Ju Chou,
Academia Sinica, Taiwan
Hwai-Jong Cheng,
University of California, Davis,
United States
*Correspondence:
Masayuki Masu
mmasu@md.tsukuba.ac.jp
†These authors have contributed
equally to this work
Received: 26 September 2019
Accepted: 27 December 2019
Published: 21 January 2020
Citation:
Aizawa S, Okada T,
Keino-Masu K, Doan TH,
Koganezawa T, Akiyama M,
Tamaoka A and Masu M (2020)
Abnormal Pyramidal Decussation
and Bilateral Projection of the
Corticospinal Tract Axons in Mice
Lacking the Heparan Sulfate
Endosulfatases, Sulf1 and Sulf2.
Front. Mol. Neurosci. 12:333.
doi: 10.3389/fnmol.2019.00333
Abnormal Pyramidal Decussation
and Bilateral Projection of the
Corticospinal Tract Axons in Mice
Lacking the Heparan Sulfate
Endosulfatases, Sulf1 and Sulf2
Satoshi Aizawa1,2,3†, Takuya Okada1,2†, Kazuko Keino-Masu1,2, Tri Huu Doan1,4,
Tadachika Koganezawa1,4,5, Masahiro Akiyama6, Akira Tamaoka1,3 and
Masayuki Masu1,2*
1Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan, 2Department of Molecular
Neurobiology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, 3Department
of Neurology, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, 4Department
of Physiology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, 5Transborder
Medical Research Center, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, 6Environmental Biology Laboratory,
Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
The corticospinal tract (CST) plays an important role in controlling voluntary movement.
Because the CST has a long trajectory throughout the brain toward the spinal cord,
many axon guidance molecules are required to navigate the axons correctly during
development. Previously, we found that double-knockout (DKO) mouse embryos lacking
the heparan sulfate endosulfatases, Sulf1 and Sulf2, showed axon guidance defects
of the CST owing to the abnormal accumulation of Slit2 protein on the brain surface.
However, postnatal development of the CST, especially the pyramidal decussation and
spinal cord projection, could not be assessed because DKO mice on a C57BL/6
background died soon after birth. We recently found that Sulf1/2 DKO mice on a
mixed C57BL/6 and CD-1/ICR background can survive into adulthood and therefore
investigated the anatomy and function of the CST in the adult DKO mice. In Sulf1/2 DKO
mice, abnormal dorsal deviation of the CST fibers on the midbrain surface persisted
after maturation of the CST. At the pyramidal decussation, some CST fibers located
near the midline crossed the midline, whereas others located more laterally extended
ipsilaterally. In the spinal cord, the crossed CST fibers descended in the dorsal funiculus
on the contralateral side and entered the contralateral gray matter normally, whereas the
uncrossed fibers descended in the lateral funiculus on the ipsilateral side and entered
the ipsilateral gray matter. As a result, the CST fibers that originated from 1 side of the
brain projected bilaterally in the DKO spinal cord. Consistently, microstimulation of 1 side
of the motor cortex evoked electromyogram responses only in the contralateral forelimb
muscles of the wild-type mice, whereas the same stimulation evoked bilateral responses
in the DKO mice. The functional consequences of the CST defects in the Sulf1/2 DKO
mice were examined using the grid-walking, staircase, and single pellet-reaching tests,
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Aizawa et al. Corticospinal Dysgenesis in Sulf1/2 Mutants
which have been used to evaluate motor function in mice. Compared with the wild-type
mice, the Sulf1/2 DKO mice showed impaired performance in these tests, indicating
deficits in motor function. These findings suggest that disruption of Sulf1/2 genes leads
to both anatomical and functional defects of the CST.
Keywords: heparan sulfate, Sulfatase 1, Sulfatase 2, knockout mouse, corticospinal tract, pyramidal decussation,
motor movement, bilateral projection
INTRODUCTION
The corticospinal tract (CST) plays a critical role in controlling
voluntary movement (Lemon, 2008;Welniarz et al., 2017a). It
is the longest tract in the central nervous system, originating
in layer 5 pyramidal neurons in the sensorimotor cortex and
terminating in the spinal cord. In rodents, the CST fibers pass
through the internal capsule and cerebral peduncle, extend
onto the ventral medulla, cross the midline at the pyramidal
decussation dorsally, and further descend in the dorsal funiculus
of the spinal cord contralaterally. Before reaching the spinal cord,
they send collateral branches to the striatum, superior colliculus,
red nucleus, pontine gray nucleus, and dorsal column nuclei
(Wang et al., 2018). In the spinal cord, the CST fibers project
to the dorsal and intermediate portions of the gray matter and
innervate interneurons, which in turn control motor neurons
(Lemon, 2008;Welniarz et al., 2017a).
Because the CST axons extend throughout the brain during
development, a number of axon guidance molecules are required
to navigate them to their targets correctly (Canty and Murphy,
2008;Leyva-Díaz and López-Bendito, 2013;Welniarz et al.,
2017a). For example, Slit and its receptor Robo guide the
axons by preventing them from entering the hypothalamic
area and crossing the midline. Netrin-1 and its receptors
DCC/Unc5, Sema6A and its receptors Plexin A3/A4, and the cell
adhesion molecules L1 and NCAM are implicated in forming
the pyramidal decussation (Dahme et al., 1997;Cohen et al.,
1998;Fransen et al., 1998;Finger et al., 2002;Rolf et al., 2002;
Faulkner et al., 2008;Rünker et al., 2008). In the spinal cord,
Wnt and its receptor Ryk promote caudal growth (Liu et al.,
2005), whereas ephrin and its receptor Eph are involved in
topographical branching and innervation (Dottori et al., 1998;
Kullander et al., 2001a,b;Yokoyama et al., 2001).
The heparan sulfate endosulfatases, Sulfatase 1 (Sulf1) and
Sulfatase 2 (Sulf2), are extracellular enzymes that remove sulfate
groups at the 6-Oposition of the glucosamine in heparan sulfate
(HS) (Lamanna et al., 2007). Because Sulf-mediated desulfation
occurs mainly in the highly sulfated regions of HS, which are
required for interaction with signaling molecules, Sulfs can
Abbreviations: ABC, avidin-biotin-peroxidase complex; AP, anterior-posterior;
BDA, biotinylated dextran amine; CMM, congenital mirror movement; CST,
corticospinal tract; DAB, 3,30-diaminobenzidine; DF, dorsal funiculus; DKO,
double knockout; DMSO, dimethyl sulfoxide; DV, dorsal-ventral; EMG,
electromyogram; HS, heparan sulfate; i.p., intraperitoneal; KO, knockout; LF,
lateral funiculus; M1, primary motor cortex; ML, medial-lateral; PBS, phosphate-
buffered saline; PBT, PBS with 0.1% Tween-20; PFA, paraformaldehyde; PKCγ,
protein kinase C gamma; ROI, region of interest; Sulf1, Sulfatase 1; TBS, Tris-
buffered saline; TBST, Tris-buffered saline supplemented with 0.1% Tween-20;
TSA, tyramide signal amplification.
regulate cellular signaling positively or negatively. Previously,
we showed that Sulf1/2 double-knockout (DKO) mice have
defects in the CST, whereas Sulf1 or Sulf2 single-knockout (KO)
mice appear normal (Okada et al., 2017). Mechanistically, in
Sulf1/2 DKO mice, abnormal accumulation of Slit2 protein in
the basement membrane of the ventral brain surface, which is
caused by the increase in 6-O-sulfated HS, results in aberrant
dorsal deflection of the CST axons on the lateral surface of
the midbrain. Most of the axons that extend abnormally in
the dorsal direction return to the medulla, whereas a part of
them enter the superior and inferior colliculi. In the medulla,
the CST fibers are defasciculated and positioned more laterally
when compared with those of controls. Because DKO mice on
a C57BL/6 background die within a day of birth, we could
not assess the postnatal development of the CST. Specifically, it
remained unclear whether pyramidal decussation and spinal cord
projection occur normally.
Recently we found that for unknown reasons, Sulf1/2 DKO
mice survived into adulthood after outcrossing to the CD-1/ICR
strain. Thus, we examined the CST trajectory in the adult Sulf1/2
DKO brain using mice on a mixed genetic background. We
found that the abnormal dorsal projection of the CST fibers
on the midbrain surface that is observed in the embryonic
brain is present in the adult brain. In addition, the DKO mice
have abnormal pyramidal decussation and aberrant bilateral
projections in the spinal cord. Consistently, stimulation of 1 side
of the motor cortex evokes bilateral responses in the forelimb
muscles of the DKO mice. Furthermore, we found that the DKO
mice perform poorly in skilled reaching and grasping tasks,
indicating that they have impaired motor movements.
MATERIALS AND METHODS
Mice
Sulf1 and Sulf2 KO mice were generated using homologous
recombination in 129/Ola-derived ES cells and maintained on
a C57BL/6N background by mating offspring of mice that had
been backcrossed to C57BL/6N for 5 successive generations (N5
generation), as described previously (Nagamine et al., 2012).
These mice were mated with the outbred CD-1/ICR strain (F1
generation), and the offspring was further mated with the N5
generation of C57BL/6N (F2 generation). In this study, offspring
of F1 or F2 on a mixed genetic background of C57BL/6N and
CD-1/ICR (;50%:50% or 75%:25%) were used. The genotypes
were determined by PCR of genomic DNAs isolated from the
tails. Wild-type and DKO mice (both from the mixed genetic
background) were used, unless otherwise stated. All animal
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experiments were approved by and performed according to
the guidelines of the Animal Care and Use Committee of the
University of Tsukuba.
Preparation of the Brain and Spinal Cord
Sections
Mice were deeply anesthetized by intraperitoneal (i.p.) injection
of an excess amount of pentobarbital and transcardially perfused
with 4% paraformaldehyde (PFA) in phosphate-buffered saline
(PBS). The brain and spinal cord were dissected and immersed
in the same fixative overnight at 4◦C. After being rinsed 3 times
in PBS, the tissues were immersed in 20% sucrose in PBS for
cryoprotection and embedded in Tissue-Tek O.C.T. compound
(Sakura Finetek Japan, Tokyo, Japan). Coronal sections (50 µm
thick) were cut on a cryostat (CM 1850; Leica Biosystems,
Wetzlar, Germany), and the sections were collected in PBS. Free-
floating sections were washed three times in PBS and once in
PBS with 0.1% Tween-20 (PBT) and serially dehydrated through
25–80% methanol/PBT and then 80% methanol/20% dimethyl
sulfoxide (DMSO). To block endogenous peroxidase activity, the
sections were incubated in 3% H2O2/80% methanol/20% DMSO
for 30 min. After rehydration, the sections were subjected to
further processing.
Immunohistochemistry
Protein kinase C gamma (PKCγ) immunohistochemistry was
done as previously described (Okada et al., 2019). Briefly, the
brain sections of mice (16–56 weeks old) were incubated twice
overnight with anti-PKCγantibody (1:200; Frontier Institute,
Hokkaido, Japan) in 0.5% blocking reagent (Roche Diagnostics,
Mannheim, Germany) in PBT at 4◦C. The sections were washed
with PBT for 15 min six times and then incubated with biotin-
conjugated anti-rabbit IgG antibody (1:600; Vector Laboratories,
Burlingame, CA, United States) in 0.5% blocking reagent in
PBT for 2 h. After being washed with PBT for 15 min five
times and with Tris-buffered saline (TBS) supplemented with
0.1% Tween-20 (TBST) for 15 min, the sections were incubated
with avidin-biotin peroxidase complex (ABC; Vectastain Elite
ABC kit; Vector Laboratories) for 30 min. The sections were
washed with 1% Tween-20/TBS for 20 min, and then with
TBST for 20 min twice, and were thereafter kept in the
same solution at 4◦C overnight. The sections were incubated
with 3,30-diaminobenzidine (DAB; Vector Laboratories) for
10 min. All steps were performed at room temperature unless
otherwise indicated.
Anterograde Tracing of the CST
Anterograde tracing of the CST was done using biotinylated
dextran amine (BDA) as follows. Adult mice of either sex (9–
45 weeks old, 17.1–53.3 g) were anesthetized by i.p. injection of
pentobarbital (50 mg/kg body weight), and placed in a stereotaxic
frame (David Kopf Instruments, Tujunga, CA, United States).
After the scalp was incised, a burr hole was made using a dental
drill. After the dura was removed, a Neuros syringe (model
75RN; Hamilton Laboratories, Reno, NV, United States) was
inserted into the target area in the right sensorimotor cortex. The
stereotaxic coordinates (anterior-posterior [AP] to the bregma;
medial-lateral [ML] from the midline; dorsal-ventral [DV] from
the pial surface, in mm) were +1.2 AP, +1.5 ML, 0.7 DV for the
forelimb area, and −1.2 AP, +1.0 ML, 0.7 DV for the hindlimb
area. Systematic injection to the sites at +1.2, 0, and −1.2 AP
and from 0.5 to 3.0 ML was also performed. BDA (10,000 MW,
lysine-fixable; Molecular Probes, Eugene, OR, United States) was
dissolved in PBS (10%) and 0.5 µl was injected over 5 min. Five
min after the injection was completed, the tip was slowly removed
from the cortex, and the mice were returned to their cages.
To examine the labeled fibers in the brain and spinal cord,
the mice were transcardially perfused with 4% PFA/PBS at 8
and 15 days after the injection, respectively. The brain sections,
prepared as described previously, were incubated with ABC
for 30 min and subjected to the DAB reaction for 20 min
at room temperature. The spinal cord sections (approximately
C2 and L1–3 levels) were further subjected to tyramide signal
amplification (TSA). For this, after ABC treatment, the sections
were incubated with biotin tyramide (TSA Biotin system;
PerkinElmer, Waltham, MA, United States) for 10 min. After
washing with TBST, the sections were again incubated with ABC
for 30 min. After washing with TBST, the signals were visualized
by incubation with DAB for 20 min at room temperature. The
sections were mounted on MAS-coated slide glasses (Matsunami
Glass Industry, Osaka, Japan), dehydrated through an ethanol
series, and cleared in xylene, and the coverslips were mounted
using Poly-mount (Polysciences, Warrington, PA, United States).
Images were observed and recorded using microscopes (Axioplan
2; Carl Zeiss Microscopy, Jena, Germany and BZ-8000; Keyence,
Osaka, Japan). In all cases, the precise location of the BDA
injection site in the target area was confirmed by examining the
stained sections.
3D Reconstruction
3D reconstruction of the serial sections was done as described
previously (Okada et al., 2019). Briefly, the 2D images of
the serial sections were aligned using AutoAligner alignment
software (Bitplane, Zürich, Switzerland) on the basis of the shape
of the sections and the location of the signals. Stacks of the
aligned images were imported into Imaris software (Bitplane) and
transformed into 3D images.
Quantification of Labeled Fibers
To examine the distribution of the BDA-labeled fibers in the
spinal cord, the BDA signals in the spinal cord sections were
quantified using ImageJ software1. Digital images were obtained
using a microscope (Axioplan 2) with a 5 ×objective lens and
a cooled CCD camera (VB-7010, Keyence). Bright-field images
were converted to 8-bit grayscale, and the colors of the images
were subsequently inverted. The threshold of the background
signal was determined using the triangle algorithm (Zack et al.,
1977) in the Auto Threshold method of ImageJ software. The
integrated density, the sum of the values of the signals above the
threshold, was measured for each region of interest (ROI). To
quantify the signals in the descending tract, ROIs were drawn
1https://imagej.nih.gov/ij/
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to outline the contralateral dorsal funiculus (cDF) and ipsilateral
lateral funiculus (iLF) (Figure 6A) using dark-field illumination,
which can highlight the border between the white and gray
matter. Percent integrated density was calculated as the ratio of
the integrated density in each ROI to the sum of the integrated
density in the cDF and iLF of the cervical and lumbar cord
regions. To quantify the signal in the contralateral and ipsilateral
side, the gray matter was divided into right and left halves. To
quantify the signal along the dorsoventral axis, the gray matter
was divided into quarters and ROIs were drawn to outline the
dorsal quarter, intermediate half, and ventral quarter on both
sides (Figure 6A). Percent integrated density was calculated as
the ratio of the integrated density in each ROI to the sum of the
integrated density in the gray matter.
Intracortical Microstimulation and
Electromyography
Intracortical microstimulation and electromyogram (EMG)
recordings were performed using the methods reported
previously (Li and Waters, 1991;Serradj et al., 2014;Gu et al.,
2017;Ueno et al., 2018) with some modifications. Briefly, after
injection of atropine sulfate (0.05 mg/kg body weight, i.p.), the
mice were anesthetized by injection of ketamine (100 mg/kg
body weight, i.p.). During surgery, isoflurane inhalation was used
in conjunction. One-fifth the amount of ketamine was further
added when a pain-induced reflex or spontaneous movement was
observed during surgery and recording. The mouse was placed
in a stereotaxic frame (Narishige, Tokyo, Japan) and craniotomy
was performed. A polyurethane-coated coaxial microelectrode
(200 µm diameter, 50 ktip resistance; Unique Medical, Tokyo,
Japan) was inserted into the motor cortex (+0.75 AP, +1.5 ML,
0.6–1.0 DV) and 14 square-pulses of current stimulations
(200-µs duration, 3-ms intervals, 20–100 µA) were delivered
every 1 s using a pulse generator (SEN-7103M; Nihon Kohden,
Tokyo, Japan) and an isolator (SS-401J, Nihon Kohden). When
forelimb movements were observed, the EMG responses were
recorded differentially using nichrome wire electrodes (tip
0.5 mm deinsulated) inserted into the bilateral biceps and triceps
muscles with an amplifier (MEG-6108, Nihon Kohden) with low-
and high-frequency cutoffs of <150 Hz and >3 kHz, respectively.
EMG responses to 4 square-pulses of current stimulation (200-µs
duration, 3-ms intervals, every 550 ms, 20–100 µA) to the motor
cortex were recorded using an AD converter (model 1401 plus;
Cambridge Electronic Design, Cambridge, United Kingdom)
and Spike2 software (ver. 7.2; Cambridge Electronic Design). The
EMGs were rectified and averaged 500 times with triggering by
microstimulations. At the end of the experiment, electric lesions
(20 µA, 10 s) were made to mark the location of the recording
sites. The mouse was transcardially perfused with 4% PFA under
deep anesthesia and the brain was histologically examined.
Grid-Walking Test
The grid-walking test was performed essentially as described
previously (Starkey et al., 2005). Male mice (11–12 weeks old)
were placed on an elevated wire grid (32 ×20 cm square
with 11 ×11 mm grids, placed 50 cm from the floor) and
allowed to explore freely for 3 min. Behavior was recorded
using a digital camera (EX-FR100; Casio, Tokyo, Japan) at 30
frames per sec and scored later. An investigator blinded to
the mouse genotype counted the number of foot-fault errors,
which were scored when one of the limbs fell below the grid
surface (Figure 8A). Because the mice moved their forelimbs
four times more frequently than their hindlimbs in this test, we
independently analyzed the first 200 steps taken by the forelimbs
and the first 50 steps taken by the hindlimbs on both sides.
The foot-fault rate (% foot fault) was calculated by dividing the
numbers of the foot-fault errors by 200 for the forelimbs and 50
for the hindlimbs.
Staircase Test
The staircase test was performed as described previously with
minor modifications (Montoya et al., 1991;Baird et al., 2001).
A staircase apparatus for mice (model 80301) was purchased from
Melquest (Toyama, Japan). It consists of a start chamber with a
clear, hinged lid and a narrow corridor with a central platform
and a double staircase (Figure 8C0). The central platform extends
along the length of the corridor, and a removable double
staircase with 8 steps on each side can be inserted into the
space between the platform and walls. Food pellets (Dustless
Precision Pellet, 20 mg; Bioserv, Flemington, NJ, United States)
are baited in the shallow well of each step (Figure 8C), and
the mouse retrieves the pellets on either side only with the
forelimb of the same side because it cannot turn around
in the corridor.
At the beginning, male mice (11–12 weeks old) were
familiarized to the food pellets by 55 mg per g body weight per
d being placed in their cages on 3 consecutive days. On the next
day, the mice were habituated to the staircase box for 30 min with
four pellets baited along the platform as well as two pellets baited
on each step of both sides. From this day, the mice were deprived
of food for 18–20 h before the daily test session. Over the next
4 days (test session), the mice were placed in the apparatus with
single pellets baited on each well in the lower seven steps on both
sides. During the test time (30 min), the mice were allowed to
enter the corridor and reach, retrieve, and eat the pellets freely.
At the end of each session, an experimenter checked the number
and place of the remaining pellets to calculate the following values
and to evaluate skilled function. Pellets baited on the second step
were not scored because many of the mice used their tongues to
retrieve pellets (wild-type, n= 5/6; DKO, n= 6/6), but pellets were
still presented on these steps because otherwise the mice seemed
to lose their motivation to reach the pellets in the lower steps. The
“number of pellets collected” (scores from 0 to 12) was calculated
by subtracting the number of the remaining pellets from the total
number of pellets baited from the third to eighth well on both
sides, which indicated the number of pellets successfully eaten by
the mouse during the session. The “maximum distance reached”
(scores from 0 to 6) indicated the deepest well reached by the
mouse (the larger of the right or left) regardless of whether it
ate, dropped, or knocked down the pellets; scores 1–6 indicate
that the mouse reached the third to eighth well, respectively. The
“success rate” was calculated by dividing the “number of pellets
collected” by the sum of the wells reached by the mouse on both
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sides. The mouse’s behavior was recorded using a digital camera
for later analysis.
Single Pellet-Reaching Test
The single pellet-reaching test was performed as described
previously, with minor modifications (Farr and Whishaw, 2002;
Chen et al., 2014). The apparatus was a clear box made of
plexiglass (20 cm high, 15 cm deep, 8.5 cm wide, measured
from the outside, and 5 mm thick) that had three vertical slits
(13 cm high, 0.5 cm wide): one central slit on one side and two
lateral slits (2.5 cm lateral to the center) on the opposite side.
At the beginning, male mice (13–15 weeks old) were habituated
to the food pellets (Dustless Precision pellet, 20 mg; Bioserv)
for 2 days, by 55 mg per g body weight per day being placed
in their cages. On the next day, the mice were familiarized to
the apparatus with 20 pellets placed in it for 20 min. From this
day, the amount of laboratory chow was adjusted to maintain
approximately 90% of the free-feeding weight. A shaping session
started on the following day. The mice were placed in the
apparatus with a tilted food tray affixed to the front of the
center slit and filled with pellets. In this session, the mice used
both forelimbs to reach for the pellets. The shaping session was
finished when the mouse performed 20 reaching attempts within
20 min. If the mouse did not perform 20 reaching attempts
within 20 min, the shaping session was conducted again on
the next day. If the mouse could not complete shaping within
5 days, it was excluded from further testing (1 of 10 wild-
type mice and 1 of 11 DKO mice). The preferred limb was
determined by counting which forelimb the mice used more
frequently (>50%) in the shaping session. The test sessions
started the day after shaping and lasted for 8 days, one session
per day. A wild-type mouse died for unknown reasons after
completing the shaping session and thus was excluded from
further analysis.
In the test sessions, the mice were placed in the apparatus
with the double-slit side facing downward. A holding plate
(10 mm high) was affixed to the front wall of the apparatus.
To place a pellet at the same position consistently, two divots
were made: 6.5 mm from the front wall and 4 mm medial
to the center of each lateral slit. A single pellet was baited
on the divot of the preferred side of each mouse. Because
the mice pronated the forelimb medially to reach for the
pellet, the medial displacement of the divot encouraged them
to reach with the preferred limb. A daily test session was
finished when 30 reaching attempts were performed or the time
limit of 20 min was exceeded. Behavior was recorded using a
digital camera at 30 frames per sec for later analysis. Reaching
attempts were classified into four categories: success, drop,
loss, and failure. “Success” means that the mouse successfully
grasped the pellet and brought it into its mouth. “Drop”
means that the mouse grasped the pellet and dropped it
inside the chamber before putting it into its mouth. “Loss”
means that the mouse grasped the pellet and dropped it
outside the chamber and thus could not eat it. “Failure”
means that the mouse missed, touched, or knocked the
pellet and failed to grasp it eventually. “Failure” also includes
drawing the pellet without grasping regardless of whether
the mouse ultimately brought the pellet into its mouth. The
rates for success, drop, loss, and failure were calculated by
dividing the numbers of success, drop, loss, and failure by the
respective total attempts.
To examine the reaching movement trajectories of the
forelimbs, five successful reaches of each mouse were analyzed
using the recorded video. All the mice except 1 Sulf1/2 DKO
mouse achieved at least five successful reaches during the
total test period. If a mouse achieved five or more successful
reaches on the last test day, the first five successful reaches
on the day were analyzed. If a mouse achieved less than 5
successful reaches on the last test day, successful reaches on
the previous days were included for analysis. The positions of
the distal tip of the second digit, second metacarpophalangeal
joint, and wrist were marked separately in single frames and
each trajectory was calculated using MTrackJ2(Meijering et al.,
2012), a plugin for ImageJ software. To analyze the velocity
profiles of the reaching movements, the positions of the distal
tip of the second digit in the five consecutive frames (P1–
P5) in a successful reach, in which P3corresponds to the
points of pellet grasping, were marked in the video, and the
velocity between 2 points (vimeans the velocity between Piand
Pi+1) was measured.
Rotarod Test
The rotarod test was performed using a rotarod apparatus (ENV-
577M; Med Associate Inc, Fairfax, VT, United States). Five mice
were put on a rod and the length of time that each remained on
the rotating rod was measured. The mice were subjected to a total
of 6 sessions on consecutive days (three sessions per day), with
accelerating speed (4–40 rpm) over 5 min.
Open Field Test
The open field test was performed using a square arena [OF-
36(M)SQ; 500 ×500 mm, wall height of 400 mm; Muromachi
Kikai, Tokyo, Japan] and a video tracking system (ANY-maze;
Stoelting, Wood Dale, IL, United States). Each mouse was allowed
to walk in the arena freely for 30 min, and the total distance
traveled was measured.
Hot Plate Test
The hot plate test was performed as previously described, with
minor modifications (O’Callaghan and Holtzman, 1975). A male
mouse (17–18 weeks old) was placed on a hot plate maintained
at 55◦C (FHP-450; Tokyo Garasu Kikai, Tokyo, Japan), and the
latency to jump, paw-shaking or paw-licking was recorded.
Statistical Analysis
All the statistical tests were performed using Prism 4.0c
(GraphPad Software; San Diego, CA, United States). Statistical
significance in the staircase test, single pellet-reaching task, and
BDA tracing study was evaluated using one-way or two-way
repeated-measures analysis of variance (ANOVA) or two-way
ANOVA with Bonferroni post hoc tests. Statistical significance
2https://imagescience.org/meijering/software/mtrackj/
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in the grid walking test, hot plate test, and velocity analysis was
evaluated using the Mann–Whitney Utest.
RESULTS
CST Defects in the Adult Sulf1/2 DKO
Brain Revealed by PKCγStaining
To examine the CST trajectory in the adult brain, we first
performed immunohistochemistry for PKCγ, a marker for CST
fibers in the adult mouse brain (Mori et al., 1990;Ding et al., 2005;
Joshi et al., 2008) because this staining is useful to overview all
the CST fibers. In the wild-type mice, the CST fibers formed the
cerebral peduncle in the caudal forebrain (Figure 1A1), passed
ventrally (Figures 1B1,C1), and extended medially toward the
pons (Figures 1D1–F1). In the medulla, the CST fibers formed
the pyramidal tract (Figure 2A1), extended toward the midline
(Figure 2B1), decussated (Figures 2C1,D1), and entered the
contralateral dorsal funiculus of the spinal cord (Figure 2E1). In
the DKO mice, the CST fibers appeared almost normal up to
the ventral midbrain (Figures 1A2–C2), whereas at the level of
the pons, the abnormal fibers extended dorsally on the surface
of the midbrain (Figures 1D2–F2). In addition, a small number
of the fibers projected toward the superior colliculus through the
thalamus (Figures 1B2–E2, 1B0
2–E0
2, arrowheads). In the medulla,
the pyramidal tract became flattened and laterally widened
(Figures 2A2,B2). A part of the fibers near the midline crossed
the midline, whereas others located more laterally extended to the
ventrolateral surface of the medulla (Figures 2C2–E2).
To examine the midline crossing, we reconstructed 3D
images of the CST trajectory from the PKCγ-stained brain
sections. In the wild-type mice, the CST fibers ran close to the
midline and crossed contralaterally at the pyramidal decussation
(Figures 2F–H). Most of the fibers entered the dorsal funiculus,
whereas a few fibers, which form the dorsolateral CST (Steward
et al., 2004), descended in a more lateral position (Figures 2E1,H,
FIGURE 1 | PKCγstaining images of the adult brain. (A–F) Coronal sections of wild-type (A1–F1)and Sulf1/2 DKO (A2–F2)brains through the cerebral peduncle
(cp) to the pons are shown. The positions of sections (A–F) in the brain are shown in the upper panel. The open and filled arrowheads indicate the normal and
abnormal projections of CST fibers, respectively. In the Sulf1/2 DKO brain, a small number of fibers projected abnormally through the thalamus to the midbrain
(B0
2–E0
2, showing magnified images in the boxed regions in B2–E2, respectively). In the Sulf1/2 DKO brain, misdirected fibers were found on the surface of the
midbrain (E2–F2)and the pyramidal tract was thinner and broader (F2, bracket) than that in the wild-type control. Aq, aqueduct; Cb, cerebellum; Cx, cerebral cortex;
f, fornix; Hi, hippocampus; MGN, medial geniculate nucleus; MN, mammillary nucleus; MO, medulla oblongata; OB, olfactory bulb; PGN, pontine gray nucleus; SC,
superior colliculus; SN, substantia nigra; SpCd, spinal cord. The scale bars indicate 1.0 mm (A1–F1,A2–F2)and 500 µm(B0
2–E0
2).
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FIGURE 2 | PKCγstaining images and their 3D reconstruction. (A–E) Coronal sections of wild-type (A1–E1)and Sulf1/2 DKO (A2–E2)brains from the medulla to the
anterior spinal cord are shown. The positions of coronal sections (A–E) in the brain are shown in the upper panel. The open arrowheads in (A–E) indicate the normal
projections of CST fibers. (F–H,J–L) 3D reconstruction of PKCγ-positive fibers. Lateral (F,J), frontal (G,K), and ventral (H,L) views of the 3D images are shown. The
CST fibers formed a pyramidal tract (py) on the ventral surface of the medulla. At the pyramidal decussation (pyx), most fibers entered the contralateral dorsal
funiculus (df), whereas a small portion descended laterally to the dorsal funiculus as the dorsolateral CST (dlcst; A1–E1,F–H). In the Sulf1/2 DKO brain, the pyramidal
tract became thinner and broader (A2–D2, brackets). The pyramidal tract gradually split into medial and lateral bundles (K,L). The medial bundle (white brackets,
K,L) entered the contralateral dorsal funiculus, whereas the lateral bundle (yellow brackets, J–L) extended on the ventrolateral surface of the ipsilateral spinal cord.
The yellow and white dotted lines in (F–H,J–L) indicate the midline and contour of the brain, respectively. Anterior-posterior (A-P) and dorsal-ventral (D-V) body axes
are shown. (I,M) Illustrate the ventral views of the CST fibers and their distance from the midline (dotted lines). The scale bars indicate 600 µm(A–E) and 1.0 mm
(F–H,J–L).
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dlcst). In contrast, the Sulf1/2 DKO mice showed defects in
the pyramidal decussation (Figures 2J–L and Supplementary
Figure S1). First, the CST fibers were located more laterally
than those in the wild-type mice: the largest distances between
the midline and the medial border of the CST fibers in the
medulla were 161.1 ±18.7 µm in the wild-type mice (n= 6 CSTs
from three mice; Figure 2I) and 414.1 ±17.6 µm in the DKO
mice (n = 6 CSTs from three mice; Figure 2M), whereas those
between the midline and the lateral borders of the CST fibers were
693.0 ±14.2 µm in the wild-type mice and 1212.6 ±28.1 µm in
the DKO mice (Figures 2I,M). Secondly, in the DKO mice, the
laterally deviated fibers descended ipsilaterally without midline
crossing, whereas the medially located fibers crossed the midline
(Figures 2K,L). The outermost fibers that crossed the midline in
the DKO mice were 765.3 ±36.9 µm apart from the midline
(Figure 2M), which was close to the distance of the outermost
fibers from the midline in the wild-type mice.
BDA Tracing of the CST Fibers in the
Sulf1/2 DKO Brain
Given that PKCγstaining is also positive in non-CST neurons,
to see the CST trajectory more specifically, we next performed
anterograde tracing of the CST fibers using BDA in wild-type
(n= 15) and Sulf1/2 DKO (n= 21) mice. BDA was stereotaxically
injected into the primary motor area and subsequently the CST
trajectories were visualized by detecting the BDA-positive fibers
by means of the avidin-biotin peroxidase complex (ABC) and
DAB reaction. In both the wild-type and the DKO brains,
the labeled fibers extended into the internal capsule and
cerebral peduncle, with their branches projecting to the striatum,
thalamus, red nucleus, and pretectum (Figures 3A1–C1,A2–C2).
At the level of the pons, in the wild-type mice, the CST
fibers turned medially from the lateral surface of the brain
(Figure 3D1), ran beneath the pons, sent branches to the pons
(Figure 3E1, open arrow), and descended caudally to form
the pyramidal tract (Figure 3F1). In the DKO mice, abnormal
CST fibers were found on the lateral surface of the midbrain
(Figures 3D2–F2). In the caudal pons, a flattened and widened
pyramidal tract was observed (Figure 3F2).
In the tectum of the wild-type mice, BDA-positive collaterals
were observed in the superior colliculus (Figure 3F1). These
fibers ran through the thalamus and formed a dense clump at
the lateral aspect of the superior colliculus (Figures 3F0
1,F00
1) and
subsequently projected medially into the intermediate layer of
the superior colliculus (Figure 3F0
1). In the DKO brain, a similar
distribution of the labeled fibers in the intermediate layer was
observed, although the density of the fibers was much higher
than in the wild-type mice (Figures 3F2,F0
2,F00
2). In addition, a
thick bundle of the labeled fibers traversed the deep layer of the
superior colliculus (n= 12/21, Figures 3F0
2,F00
2).
In the medulla of the wild-type mice, the labeled fibers crossed
the midline (Figures 3H1,H0
1,I1). In the DKO mice, the labeled
fibers split into 2 bundles. The lateral bundle extended in the
ventrolateral position ipsilaterally (n= 21/21; Figures 3G2–I2,
black arrowheads), whereas the medial bundle reached the
midline at the pyramidal decussation and most of the fibers
crossed the midline (Figures 3H2–I2, open arrowheads). In some
cases, a few fibers reached the midline and returned to the
ipsilateral side (n= 8/21; Figures 3H2–I2, blue arrowheads);
projection to the ipsilateral dorsal funiculus was also observed
in the wild-type mice (n= 2/15, data not shown), but the
frequency and number of these fibers were higher in the
DKO mice. Other fibers entered the ventral funiculus on the
ipsilateral side in the DKO mice (n= 9/21; Figure 3H0
2, red
arrowhead); a few fibers were also seen in the ipsilateral ventral
funiculus of the wild-type mice (n= 2/15, data not shown).
These analyses clearly demonstrated the presence of midline
crossing errors in the Sulf1/2 DKO mouse (see Supplementary
Table S1 for summary).
Next, we performed 3D reconstruction of the BDA-labeled
CST fibers in the whole brain. In the wild-type mice, the
CST fibers descended through the internal capsule, cerebral
peduncle, and pyramidal tract and crossed the midline at the
pyramidal decussation (Figures 4A,B). It was apparent that the
CST fibers entered the contralateral dorsal funiculus (Figure 4B).
In addition to the main tract, collateral branches and their fiber
terminals were observed in the striatum, thalamus, superior
colliculus, pons, and dorsal column nuclei (Figure 4A;Catsman-
Berrevoets and Kuypers, 1981;O’Leary et al., 1990;Lévesque
et al., 1996;Wang et al., 2018). In the DKO mice, the most
prominent abnormality was the U-shaped trajectory in the
midbrain (n= 21/21; Figures 4C,E; arrows). After the fibers
extended dorsally toward the superior colliculus, most of them
returned to the pons. These data clearly showed that the aberrant
U-shaped detour of the CST fibers observed in the embryonic
DKO brain persisted into adulthood in all the DKO mice. In
the 3D reconstruction images, variable decussation defects were
observed in the DKO mice. Consistent with the PKCγstaining
results, the lateral bundle extended ipsilaterally (Figures 4D,F,
white arrowheads), whereas the medial bundle reached the
midline and some of the fibers crossed the midline (Figures 4D,F,
open arrowheads) and others returned to the ipsilateral side
(Figure 4D, blue arrowhead). Furthermore, defasciculation of the
CST fibers in the cerebral peduncle was also observed (n= 9/21;
Figure 4E, arrowheads). Information about all the mice analyzed
and their phenotypes is summarized in Supplementary Table S1.
Bilateral CST Fiber Projections in the
Spinal Cord of Sulf1/2 DKO Mice
Next, we examined the projection of the CST fibers in the
spinal cord. When BDA was injected stereotaxically into the
forelimb area of the Primary motor cortex (M1) of the wild-
type mice (Figure 5A; a representative stereotaxic coordinate,
+1.2 AP, +1.5 ML, 0.7 DV, in mm), the labeled fibers were
observed in the ventromedial portion of the dorsal funiculus in
the cervical spinal cord on the contralateral side of the injection
(Figure 5C). The fibers entered the intermediate portion of the
gray matter (Figure 5C) and their terminals showed bouton-like
structures (Figures 5C0,C00, open arrows). Thus, the CST fiber
projection was almost exclusively confined to the contralateral
side in the wild-type mice. Few signals reached the lumbar spinal
cord (Figure 5D).
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FIGURE 3 | BDA tracing of CST fibers. (A–I) Coronal sections of BDA-injected wild-type (A1–I1)and Sulf1/2 DKO (A2–I2)brains are shown. The positions of
sections (A–I) in the brain are shown in the upper panel. The open and filled arrowheads indicate the normal and abnormal projections of CST fibers, respectively.
The open arrows indicate collateral branches. The dotted lines indicate the midline. (F0
1,F0
2,F00
1,F00
2,H0
1,H0
2)show magnified images in the boxed regions in
(F1,F2,F0
1,F0
2,H1,H2), respectively. (A1,A2)Illustrate BDA injection sites in the motor cortex. The labeled CST fibers passed through the internal capsule (ic) and
cerebral peduncle (cp) with collateral branches projecting to the pretectum (Pt) and red nucleus (RN) in both the wild-type and the Sulf1/2 DKO brains
(B1,B2,C1,C2). In the Sulf1/2 DKO brain, the aberrant fibers were found on the surface of the midbrain (D2–F2). In the DKO medulla, some fibers reached the
pyramidal decussation (pyx), whereas others deviated laterally and descended ipsilaterally in the ventrolateral position (G2–I2, black arrowheads). At the pyramidal
decussation, some fibers crossed the midline normally and entered the contralateral dorsal funiculus (G2,H0
2,I2; open arrowheads). A small portion of fibers turned
dorsally but did not cross the midline and entered the ipsilateral dorsal funiculus (H0
2,I2; blue arrowheads). Other aberrant fibers descended in the ipsilateral ventral
funiculus (H0
2; red arrowhead). DCN, dorsal column nuclei; IC, inferior colliculus; PGN, pontine gray nucleus; py, pyramidal tract; SC, superior colliculus; Str, striatum;
Th, thalamus. The scale bars indicate 1.0 mm (A1–C1,A2–C2), 850 µm(D1–F1,D2–F2), 300 µm(F0
1,H0
1,F0
2,H0
2), 500 µm(G1–I1,G2–I2), and 100 µm(F00
1,F00
2).
In the DKO mice, the labeled fibers descended in the lateral
funiculus on the ipsilateral side in addition to in the dorsal
funiculus on the contralateral side (Figure 5G). The fibers in
the lateral funiculus entered the ipsilateral gray matter in a
medial direction (Figure 5G0, arrowheads), whereas the fibers
in the dorsal funiculus entered the contralateral gray matter
in a ventrolateral direction (Figure 5G). In addition, a few
fibers crossed the midline in the spinal cord (Supplementary
Figure S2). The terminals of the fibers from both the lateral
and the dorsal funiculi had bouton-like structures (Figure 5G00,
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FIGURE 4 | 3D images of the BDA-labeled CST. (A–F) Representative images
from the wild-type (A,B) and 2 Sulf1/2 DKO (C–F) mice are shown. DKO
mouse #1 (C,D) is the same as the one shown in Figure 3. Lateralviews of
the whole brain (A,C,E) and ventral views of the medulla (B,D,F) are shown.
The yellow and white dotted lines indicate the midline and contour of the
brain, respectively. The asterisks indicate BDA injection sites. In the wild-type
brain (A,B), a whole image of the CST from the motor cortex (Cx) to the
contralateral dorsal funiculus (df; open arrowhead) was successfully obtained.
DKO mice showed abnormal looping of the labeled fibers on the surface of
the midbrain (C,E; arrows). In the cerebral peduncle (cp) of Sulf1/2 DKO
mouse #2, the CST fibers were slightly defasciculated (E, filled arrowheads). In
the medulla of the DKO mice, laterally located fibers projected ipsilaterally to
the spinal cord (D,F; filled arrowheads). At the pyramidal decussation (pyx),
almost all the fibers that extended to the midline crossed the midline in Sulf1/2
DKO mouse #2 (F, open arrowhead), whereas a part of the fibers approached
the midline but entered the ipsilateral dorsal funiculus in Sulf1/2 DKO mouse
#1 (D, blue arrowhead). Anterior-posterior (A-P), dorsal-ventral (D-V), and
right-left (R-L) body axes are shown. DCN, dorsal column nuclei; ic, internal
capsule; IC, inferior colliculus; Pn, pons; py, pyramidal tract; SC, superior
colliculus; Str, striatum; Th, thalamus. The scale bars indicate 1.0 mm (A,C,E)
and 600 µm(B,D,F).
arrows). Thus, the CST fiber projection was bilateral in the DKO
mice. Few signals reached the lumbar spinal cord (Figure 5H),
indicating that the CST fibers originating from the forelimb area
projected to their inherent projection levels in the DKO mice.
When BDA was injected into the hindlimb area of the M1
of the wild-type mice (Figure 5B; a representative stereotaxic
coordinate, −1.2 AP, +1.0 ML, 0.7 DV), the labeled fibers passed
into the contralateral dorsal funiculus at the cervical level with
few projecting into the gray matter (Figure 5E). At the lumbar
level, the fibers in the dorsal funiculus projected into the dorsal
portion of the gray matter (Figures 5F,F0,F00). In the DKO
mice, the labeled fibers descended in both the ipsilateral lateral
funiculus and the contralateral dorsal funiculus (Figures 5I,J).
Projection into the gray matter was scarce in the cervical spinal
cord (Figure 5I), whereas bilateral projection to the dorsal
portion was observed in the lumbar spinal cord (Figures 5J,J0,J00).
These findings suggest that the CST fibers of the DKO mice
terminate at the appropriate levels of the spinal cord even though
the trajectories are aberrant.
To compare the differences in the CST projection between
the wild-type and DKO mice precisely, we quantitated the
distribution of the BDA-labeled fibers in the spinal cord. Because
the extent of labeling differed among individuals, we measured
the total number of signals present in the cervical or lumbar
spinal cord and calculated the percentages of the signals in the
regions of interest (ROIs). To this end, we used the integrated
density, the sum of the values of the signals above the threshold,
which was determined using ImageJ software. The ratio of the
labeled fibers in each ROI among all the fibers in the white
or gray matter was shown as the percent integrated density,
the ratio of the integrated density in each ROI to the sum of
integrated density in the white or gray matter in the cervical
or lumbar spinal cord (please refer to “Quantification of labeled
axons” in the section “Materials and Methods” for a detailed
description of the method). We first compared the percentages
of the normal fibers in the contralateral dorsal funiculus (cDF)
and of the abnormal fibers in the ipsilateral lateral funiculus
(iLF, Figure 6A). Quantitative analyses demonstrated that the
CST fibers were confined to the cDF in the wild-type mice,
whereas they were present more in the iLF and less in the cDF
in the Sulf1/2 DKO mice (Figures 6B,C). Next, we compared
the laterality. The CST fibers projected to the gray matter
on the contralateral side in the wild-type mice, whereas they
projected bilaterally in the DKO mice (Figures 6D,E). Finally,
we compared the dorsoventral distribution of the CST fibers. For
this purpose, we divided the spinal cord into four parts along
the dorsoventral axis and compared the distribution in the dorsal
quarter, intermediate half, and ventral quarter (Figure 6A). This
analysis showed that when the signals on both sides were added,
the dorsoventral distribution within the gray matter was almost
the same in the wild-type and DKO mice (Figures 6F,G).
Topographical Organization of the Motor
Area in Sulf1/2 DKO Mice
We then wondered whether the topographical organization of the
motor cortex is the same in wild-type and Sulf1/2 DKO mice.
To address this question, we performed systematic injection
of BDA at different sites throughout the sensorimotor cortex
(Supplementary Figure S3) and examined the projection in the
cervical and lumbar spinal cord regions. Dense projection to the
cervical spinal cord was found when BDA was injected at +1.2
and 0 AP, from 1.0 to 2.0 ML in both the wild-type and the DKO
mice (Supplementary Figure S3). Projection to the lumbar spinal
cord was found when BDA was injected at 0 AP, from 1.0 to
1.5 ML, and at −1.2 AP, from 0.5 to 1.5 ML in both the wild-
type and the DKO mice (Supplementary Figure S4), indicating
that the motor area projecting to the lumbar spinal cord was
caudal and medial to that projecting to the cervical spinal cord,
with overlap between them, which is consistent with the motor
cortex map determined by electrical or optogenetic stimulation
(Li and Waters, 1991;Ayling et al., 2009;Tennant et al., 2011).
Thus, the overall topographical organization of the motor area of
the wild-type and DKO mice was roughly the same. These data
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FIGURE 5 | Projection of BDA-labeled CST fibers in the spinal cord. (A,B) BDA injection sites for the forelimb (A) and hindlimb (B) areas. The red and blue dotted
lines encompass the forelimb and hindlimb areas, respectively. Stereotaxic coordinates for representative injection sites (red and blue spots) are shown on the right.
(C–J) Images of the transverse sections of the cervical and lumbar spinal cord regions of the wild-type (C–F) and Sulf1/2 DKO mice (G–J) are shown. The dashed
lines delineate the borders of the dorsal funiculus (DF) and ipsilateral lateral funiculus (LF). The solid lines indicate the midline. (C0,C00,F0,F00 ,G0,G00 ,J0,J00 )Show the
magnified pictures of the boxed area in (C,C0,F,F0,G,G0,J,J0), respectively. In the wild-type mouse, the labeled CST fibers that descended in the contralateral DF
projected to the dorsal horn and intermediate zone (C,F) and formed terminal arbors with bouton-like structures (C00,F00 ; open arrows). In the Sulf1/2 DKO mouse,
the labeled CST fibers descended in both the contralateral DF and the ipsilateral LF (G,I,J) and projected bilaterally in the gray matter (G,J). The terminal arbors in
both the contralateral (open arrows) and the ipsilateral gray matter (filled arrows) showed bouton-like structures (G00,J00 ). The arrowheads in (G0)and (J0)show the
fibers that entered the gray matter from the ipsilateral LF. The scale bar indicates 200 µm(C–J),35µm(C0,F0,G0,J0), and 20 µm(C00,F00 ,G00 ,J00 ).
also suggest that in DKO mice, the CST fibers take two routes
through the contralateral dorsal funiculus and the ipsilateral
lateral funiculus regardless of the positions of their origin in the
motor cortex and that they terminate at their inherent levels
regardless of the route they take.
M1-Stimulated Bilateral Responses in
Sulf1/2 DKO Mice
Our BDA tracing studies clearly demonstrated that the CST
fibers projected bilaterally in the spinal cord of the Sulf1/2 DKO
mice. This observation led us to wonder whether the uncrossed
fibers projecting to the ipsilateral spinal cord in the DKO
mice control motor neuron activity. To test this, we measured
the EMG responses after intracortical microstimulation of the
M1. An anesthetized mouse was placed in a stereotaxic frame
and a stimulating electrode was inserted into the M1 area.
Electrical stimulation of the M1 on one side evoked contralateral
forelimb movements in the wild-type mice, whereas the same
stimulation evoked bilateral movements in the DKO mice.
Consistent with the forelimb movements, when EMG responses
were observed, activation of the EMG after four pulses of
intracortical microstimulations was observed only from the
contralateral muscles of the wild-type mice, whereas bilateral
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FIGURE 6 | Quantitation of the BDA-labeled CST fibers in the spinal cord. (A) Regions of interest (ROIs) for quantifying the BDA-labeled fibers. The ROIs for the
contralateral dorsal funiculus (cDF) and ipsilateral lateral funiculus (iLF) (outlined by gray lines); contralateral (Contra) and ipsilateral (Ipsi) gray matter (outlined by
orange lines); and dorsal (D), intermediate (Int), and ventral (V) gray matter (outlined by green lines) are shown. Representative images of the cervical spinal cord from
wild-type and Sulf1/2 DKO mice that received BDA injection in the forelimb area (the same as the ones shown in Figures 5C,G) are shown. The scale bar indicates
200 µm. (B–G) The integrated density, the sum of the values of the signals above the threshold, was measured for each ROI, and its rate to the sum of the
integrated density in all the ROIs analyzed in the cervical and lumbar spinal cord regions is shown as a percentage. The percent integrated density of the BDA signals
after injection into the forelimb (B,D,F) and hindlimb (C,E,G) areas in the wild-type (n= 6) and DKO (n= 6) mice is plotted. In (F,G), the integrated densities on both
sides are combined and analyzed. Data shown are means ±SEMs. Statistical significance was calculated using two-way ANOVA with a Bonferroni post hoc test
(*P<0.05, **P<0.01, ***P<0.001).
responses were detected in the DKO mice (Figure 7 and
Supplementary Figure S5). These results show that the CST
fibers are functionally connected to both the ipsilateral and the
contralateral motor neurons, thereby controlling bilateral motor
neuron activity in the DKO mouse.
Deficits in Fine Motor Movements in
Sulf1/2 DKO Mice
The Sulf1/2 DKO mice appeared to behave normally: they did
not show gait disturbance, ataxia, or abnormal movements.
Consistent with this, the DKO mice performed normally in the
rotarod and open field tests (Supplementary Figures S6A,B),
suggesting that gross motor movements were normal in the
DKO mice. In addition, in the hot plate test, the wild-
type and DKO mice showed no significant differences in the
time taken to avoid painful thermal stimuli, indicating that
temperature and pain sensation was normal in the DKO mice
(Supplementary Figure S6C). To examine possible deficits in
fine motor movements in Sulf1/2 DKO mice, we performed
three different behavioral tests that have been used for evaluating
the motor functions of normal mice and disease models
(Brooks and Dunnett, 2009;Schönfeld et al., 2017). First, we
performed a grid-walking test that can assess sensorimotor
function and motor coordination (Starkey et al., 2005). In this
test, the mice were made to walk on an elevated wire grid for
3 min and foot-fault errors were analyzed (Figure 8A). The rate
for the foot fault was higher in the Sulf1/2 DKO mice than in the
wild-type mice: the difference was significant in the hindlimbs but
not in the forelimbs (Figure 8B).
We next performed a double staircase test (Montoya et al.,
1991;Baird et al., 2001). To this end, we used an apparatus that
has a start chamber and a narrow corridor with a central platform
and a double staircase (Figure 8C0). The removable staircase has
eight steps on each side, and small food pellets are placed in
the shallow well of each step (Figure 8C). In this apparatus, a
mouse can retrieve pellets on either side using the forelimb of the
same side (Figure 8D, arrows), enabling evaluation of the mouse’s
ability to reach the pellets with the respective forelimbs. A food-
deprived mouse was placed in the apparatus with single pellets
baited on each well on both sides, and the mouse was allowed
to retrieve pellets freely for 30 min over 4 days. At the end of
each session, the number and place of the remaining pellets were
examined. The number of pellets that the mouse ate successfully
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FIGURE 7 | Motor-evoked potentials in the forelimb muscles. (A,B)
Representative EMG responses in the triceps muscle in the wild-type (A) and
DKO mice (B). Responses of EMGs in bilateral triceps muscles to four
square-pulses of current stimulations (200-µs duration, 3-ms intervals, every
550 ms, 50–100 µA) to the M1 cortex were recorded. The EMGs were
rectified and averaged 500 times. M1 stimulation on one side evoked motor
potentials only in the contralateral triceps muscles in the wild-type mice,
whereas the same stimulation evoked bilateral responses in the Sulf1/2 DKO
mice. The asterisks indicate stimulation artifacts.
(“number of pellets collected”) was lower in the DKO mice than
in the wild-type mice (Figure 8E). The “success rate,” which was
calculated by dividing the “number of pellets collected” by the
sum of the wells reached by the mouse on both sides (Figure 8F),
was also lower in the DKO mice than in the wild-type mice
throughout the test period (Figure 8G), suggesting motor deficits
in forelimb movement.
We then performed a single pellet-reaching test (Farr and
Whishaw, 2002;Chen et al., 2014). In this test, the mice were
trained to use a forelimb to retrieve a small pellet through a
narrow slit of a clear box (Figures 9A,B). First, a food-deprived
mouse was trained to retrieve pellets from the center slit until
the mouse performed 20 reaching attempts within 20 min.
Because the mouse used both forelimbs, the preferred limb was
determined. In the subsequent test sessions (one session per
day, for 8 days), a single pellet was placed in a divot at the
fixed position (Figure 9B) to force the mouse to retrieve the
pellet with the preferred forelimb. The rate at which the mouse
successfully grasped a pellet and brought it into its mouth was
higher in the wild-type mice than in the DKO mice (Figure 9C),
indicating impaired performance in pellet retrieval in the DKO
mice. The success rate became higher during the test sessions
in the wild-type mice but not in the DKO mice (Figure 9C and
Supplementary Figure S7).
To examine why the DKO mice were less successful at
reaching the pellets, we performed kinematic measures of the
trajectory and velocity of the forelimb movement (Whishaw,
1996). The positions of the distal tip of the second digit, second
metacarpophalangeal (MCP) joint, and wrist were marked
separately in single frames and each trajectory was examined
(Figure 9D). As shown in Figure 9E, the trajectories of the digit
and MCP joint, but not of the wrist, in the successful reaches were
shorter in the DKO mice than in the wild-type mice. In addition,
the speed of the digit near the pellet was slowed down in the
wild-type mice but accelerated in the DKO mice (Figures 9F–K).
This analysis revealed that DKO mice use different goal-directed
action strategies even in successful reaches.
DISCUSSION
In this study, by taking advantage of the survival into adulthood
of Sulf1/2 DKO mice on a mixed genetic background, we
demonstrated several aspects of the CST defects in the adult
brain. First, the abnormal dorsal extension of the CST fibers
toward the superior colliculus, which was observed in DKO
embryos (Okada et al., 2017), persisted in the adult brain.
Second, at the pyramidal decussation, some fibers located close
to the midline crossed the midline, whereas others located more
laterally did not. Third, in the spinal cord, the crossed fibers
descended in the contralateral dorsal funiculus and entered the
contralateral gray matter, whereas the uncrossed fibers descended
in the lateral funiculus on the ipsilateral side and entered
the ipsilateral gray matter. These results showed that the CST
fibers of Sulf1/2 DKO mice project bilaterally in the spinal
cord. Consistently, electric stimulation of M1 neurons on 1
side evoked bilateral EMG responses in the DKO mice. We
also demonstrated impaired performance of the DKO mice in
behavioral tests used for evaluating motor functions, suggesting
deficits in their motor movement.
Pyramidal Decussation Defects
Various CST defects at the pyramidal decussation were reported
in mutant mice (Canty and Murphy, 2008;Leyva-Díaz and
López-Bendito, 2013;Welniarz et al., 2017a). These mutant mice
include mutants for Semaphorin 6A (Sema6a) and its receptors
Plexin A3 (Plxna3)/Plexin A4 (Plxna4; Faulkner et al., 2008;
Rünker et al., 2008); Netrin receptors DCC (Kanga mutant
expressing a truncated DCC protein [Dcckanga]) and UNC5C
(rostral cerebellar malformation mutant, Unc5crcm;Finger et al.,
2002); and cell adhesion molecules NCAM (Ncam1;Rolf et al.,
2002) and L1 (L1cam;Dahme et al., 1997;Cohen et al., 1998).
In the Sema6a,Plxna3, or Plxna4 KO mice and the
Plxna3/Plxna4 DKO mice, a part of the CST fibers deviated
from the midline and ran down the ventrolateral position of the
medulla, whereas those near the midline decussated normally
(Faulkner et al., 2008;Rünker et al., 2008). The authors of
those studies argued that the phenotype was caused by the loss
of the constraint of the CST fibers to the midline, which is
normally brought about by the repulsive signal of Sema6A in
the inferior olive (Faulkner et al., 2008;Rünker et al., 2008). The
CST abnormalities of these KO mice seem similar to those of
the Sulf1/2 DKO mice. However, the Sema6a KO mice showed
aberrant circumferential projection of the CST fibers in the
medulla (Okada et al., 2019), which was never observed in the
Sulf1/2 DKO mice, indicating that the molecular mechanisms
causing the decussation defects differ for Sema6a-Plxna3/a4 and
Sulf1/2 mutant mice.
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FIGURE 8 | Grid-walking and staircase tests. (A,B) Grid-walking test. When one of the limbs fell below the grid surface during free walking on the grid (A, arrow), it
was counted as a foot-fault error. (B) The foot-fault errors during the first 200 steps taken by the forelimbs and the first 50 steps taken by the hindlimbs were
analyzed. The foot-fault rate was higher in the Sulf1/2 DKO mice than in the wild-type mice: the difference was significant in the hindlimbs (3.7 ±0.6 vs. 1.0 ±0.4;
n= 6; *P<0.05, Mann–Whitney Utest) but not in the forelimbs (18.6 ±1.6 vs. 14.8 ±0.4; P= 0.065). (C–G) Staircase test. (C) Double staircase separated from
the chamber. Single food pellets were placed on each well in the lower seven steps on both sides. (C0)Staircase test apparatus. The double staircase was inserted
into the space between the platform and walls. A mouse was placed into the white chamber (left) and allowed to enter the central platform (right) and retrieve pellets
for 30 min. (D) Video recording of mouse behavior. The mouse reached, grasped, and brought a pellet to its mouth successfully (arrows). When a mouse failed to
retrieve a pellet, it was moved to a different well or to the inaccessible floor (arrowheads). (E–G) Results of the double staircase test. The “number of pellets
collected” and “success rate” were lower in the Sulf1/2 DKO mice (n= 6) than in the wild-type mice (n= 6) throughout the test period (E,G). The “maximum distance
reached” indicates the deepest well reached by the mouse. Please refer to the section “Materials and Methods” for a detailed description of the analysis. Data shown
are means ±SEMs. Statistical significance was calculated using two-way repeated-measures ANOVA with a Bonferroni post hoc test (E–G; *P<0.05, **P<0.01,
***P<0.001).
In the Dcckanga mutant mice, the CST fibers spread into the
medial and lateral bundles just before the pyramidal decussation
and neither of them crossed the midline (Finger et al., 2002;
Welniarz et al., 2017b). The medial bundle extended ipsilaterally
in the ventral funiculus, whereas the lateral bundle extended
ipsilaterally in the ventral portion of the lateral funiculus (Finger
et al., 2002). Thus, the CST defects in the Dcckanga mutant
mice seem to differ from those in the Sulf1/2 DKO mice. In
the Unc5crcm mutant mice, the CST fibers formed two distinct
bundles at the pyramidal decussation: one bundle in the normal
position near the midline and another located more laterally.
Whilst the fibers of the former crossed the midline normally,
most entered the dorsal gray matter adjacent to the dorsal
funiculus instead of entering into the dorsal funiculus (Finger
et al., 2002). Thus, the properties of the crossed fibers differ from
those in the Sulf1/2 DKO mice. By contrast, the fibers of the
laterally located bundle descended ipsilaterally in the outermost
region of the lateral funiculus of the spinal cord (Finger et al.,
2002). Therefore, the decussation error phenotype in the Unc5c
mutant mice is similar to that in the Sulf1/2 DKO mice. In
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FIGURE 9 | Single pellet-reaching task. (A) Apparatus for the single pellet-reaching task. (B) In a successful reach, a mouse extends a forelimb through the slit,
grasps a pellet in a divot, and brings it to its mouth (arrows). (C) Rate of successful reaching during eight test sessions. The wild-type mice (n= 8) showed
improvements in the success rate over time [F(7,49) = 3.19, P= 0.0072], whereas the Sulf1/2 DKO mice (n= 10) showed a lower success rate throughout the test
period [F(7,63) = 0.749, P= 0.63]. As a result, the success rates of the DKO mice were lower than those of the wild-type mice after the third session. (D,E)
Trajectory analysis of the forelimb in the successful reaches. The positions of the distal tip of the second digit, second metacarpophalangeal (MCP) joint, and wrist
were marked in the video (D, blue dots) to analyze the reaching trajectories. Representative trajectories (blue lines) in the wild-type and Sulf1/2 DKO mice are shown.
The dotted lines indicate the position of the slit. The trajectory length of the digit and MCP joint was shorter in the Sulf1/2 DKO mice (n= 9) than in the wild-type mice
(n=8)(E).(F–K) Velocity analysis of the forelimb in successful reaches. The positions of the distal tip of the second digit in the five consecutive frames (P1–P5) in a
successful reach, in which P3corresponds to the points of pellet grasping, were marked in the video, and the velocity between 2 points (v1to v4) was measured (F).
In the wild-type mice, v2and v3were smaller than v1and v4, respectively (G,I–K). In contrast, in the Sulf1/2 DKO mice, v2and v3were larger than v1and v4,
respectively (H–K). Statistical significance was calculated using one-way and two-way repeated-measures ANOVA with a Bonferroni post hoc test in (C,E) and a
Mann–Whitney Utest in (I–K). *P<0.05, **P<0.01, ***P<0.001. Data shown are means ±SEMs.
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this regard, it is interesting that different types of HS selective
for DCC and UNC-5 are assumed to lead to the formation
of the DCC/DCC and DCC/UNC-5 complex with Netrin-1,
thereby causing axonal attraction and repulsion, respectively
(Finci et al., 2014). Sulf1/2-mediated modification of HS may
affect HS-dependent formation of the netrin receptor complex
and consequent netrin-mediated responses.
In the L1cam and Ncam1 KO mice, a significant portion
of the CST fibers failed to cross the midline and entered the
ipsilateral dorsal funiculus (Cohen et al., 1998;Rolf et al., 2002).
In addition, in the Ncam1 KO mice, the CST fibers frequently
extended laterally instead of growing dorsally (Rolf et al., 2002).
In the L1cam KO mice, the CST fibers were reduced in number
and not observed caudal to the cervical spinal cord (Dahme et al.,
1997;Cohen et al., 1998). These phenotypes differ largely from
those observed in the Sulf1/2 DKO mice.
These comparisons revealed that none of the mice with loss-
of-function mutations in one of these genes showed the same CST
phenotype as that of the Sulf1/2 DKO mice. It is possible that
the gain-of-function mutations in these genes are related to the
phenotype of the DKO mice or that the combination of mutations
in more than one gene causes the phenotype.
In the Sulf1/2 DKO mice, the CST fibers within approximately
700 µm of the midline decussated almost normally, whereas the
fibers outside this border did not, suggesting that the midline-
crossing signals may operate within this range. Viewed from
this standpoint, it is likely that the location of the CST fibers in
the medulla after their return from the aberrant detour in the
midbrain may determine whether they cross the midline. Lateral
positioning of the CST fibers may be influenced by Slit-Robo
signaling, as proposed in mutant Drosophila and mouse studies
(Rajagopalan et al., 2000;Simpson et al., 2000;Farmer et al., 2008;
Jaworski et al., 2010).
Descending CST Fibers in the Lateral
Funiculus of the Spinal Cord
The CST is a major descending pathway that appeared in
mammals evolutionarily. Although the anatomy of the CST is
similar across species, the location of its fibers in the spinal
cord is different from one species to another (Lemon, 2008;
Welniarz et al., 2017a). In rodents, the crossed fibers are located
in the ventral portion of the dorsal funiculus, whereas a small
number of uncrossed fibers are present in the ventral funiculus
(Brösamle and Schwab, 1997). Despite the different locations, the
crossed and uncrossed fibers originate from the same cortical
areas (Brösamle and Schwab, 1997). In primates, by contrast, the
crossed fibers are located in the dorsolateral funiculus, whereas
the uncrossed fibers are located in the ventral and dorsolateral
funiculi (Welniarz et al., 2017a). In Sulf1/2 DKO mice and in Dcc
and Unc5c mutants, the uncrossed fibers descend in the lateral
funiculus, suggesting that it provides a permissive substrate for
CST axon elongation beyond species. It will thus be interesting
to investigate the molecular mechanisms that determine the
trajectory choice, especially the genetic elements that influence
species differences, as shown in a previous study that revealed
the mechanism underlying manual dexterity (Gu et al., 2017).
In addition, our finding that the CST fibers of the Sulf1/2 DKO
mice terminate in their inherent levels irrespective of the route
they take suggests the presence of mechanisms that stop the
projection of the growing axons at the appropriate levels of
the spinal cord.
Bilateral Projection of the CST Fibers in
the Spinal Cord
In rodents, the majority of CST fibers cross the midline at
the pyramidal decussation and project to the contralateral
spinal cord (Welniarz et al., 2017a). In Sulf1/2 DKO mice, in
addition to the normal, contralateral projection of the crossed
fibers, the uncrossed fibers enter the ipsilateral spinal cord,
resulting in bilateral projection. Our EMG studies showed that
microstimulation of the M1 on 1 side evoked comparable
responses in the bilateral forelimb muscles, indicating that the
uncrossed fibers also form functional synapses and control the
ipsilateral motor activity.
Bilateral CST projection was also observed in other mutant
mice that lack ephrin B3 (Efnb3), EphA4 (Epha4), or α-chimerin
(Chn1), although the location of the abnormal midline crossing
differs between these mice and Sulf1/2 DKO mice. In the above
mutants, the CST fibers cross the midline at the pyramidal
decussation and descend in the contralateral dorsal funiculus
normally, but a considerable amount of the fibers cross the
midline again after entering the gray matter, resulting in bilateral
projection (Dottori et al., 1998;Kullander et al., 2001a,b;
Yokoyama et al., 2001;Iwasato et al., 2007). In addition to the
misprojection of the CST fibers, axons of the interneurons in the
spinal cord cross the midline abnormally as a result of the loss
of the midline barrier signal by ephrin B3. Interestingly, these
mutant mice show a hopping gait phenotype, which was thought
to be caused by the inability to perform left-right alternate
movement during locomotion (Kullander et al., 2003;Borgius
et al., 2014;Serradj et al., 2014;Katori et al., 2017). Spinal cord-
specific deletion of Epha4 or Chn1 led to a hopping gait, whereas
forebrain-specific disruption of the same genes did not lead to the
phenotype (Borgius et al., 2014;Katori et al., 2017), suggesting
that a hopping gait is caused by abnormal midline crossing of
interneurons in the spinal cord. Given that Sulf1/2 DKO mice
did not show a hopping gait, they did not have deficits in the
locomotor circuit, although a few CST fibers crossed the midline
in the spinal cord in those mice.
Interestingly, neurological conditions similar to the
anatomical and functional CST abnormalities found in the
Sulf1/2 DKO mice are observed in patients with congenital
mirror movement (CMM). CMM is a rare genetic disorder that
is characterized by involuntary movements on one side of the
body induced by intentional movements on the opposite side
(Galléa et al., 2011;Cox et al., 2012;Welniarz et al., 2017a) as the
result of mutations in the DCC,RAD51, and NTN1 (netrin-1)
genes (Srour et al., 2010;Depienne et al., 2012;Méneret et al.,
2017). In some CMM patients, the proportion of the uncrossed
CST fibers at the pyramidal decussation was increased and
unilateral M1 stimulation elicited bilateral responses (Méneret
et al., 2017;Welniarz et al., 2017b). Therefore, the patterns of
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Aizawa et al. Corticospinal Dysgenesis in Sulf1/2 Mutants
the anatomical and physiological deficits are almost the same as
those in the Sulf1/2 DKO mice, although these mice did not show
apparent mirror movement in their cage or in our behavioral
tests. However, forebrain-specific EphA4 KO mice, which did
not show a hopping gait in normal stereotypical locomotion
despite the existence of abnormal midline crossing of the CST
fibers in the spinal cord, showed abnormal synchronization of
left and right forelimb movement in adaptive locomotion over
obstacles (Serradj et al., 2014). Thus, it would be interesting
to examine whether Sulf1/2 DKO mice show synchronized
movement in the same behavioral test. It may be useful to
elucidate the contribution of midline-crossing abnormality to
mirror movement and to address the question whether midline-
crossing errors at the pyramidal decussation and in the spinal
cord have different consequences for motor control in left-right
synchronization.
Impaired Motor Movement
The CST plays an important role in cortical control of spinal
motor neuron activity (Lemon, 2008;Welniarz et al., 2017a).
Because it is a major pathway for voluntary movement, its
dysfunction leads to motor impairment. In mice, lesions of
the motor cortex, surgical dissection of the pyramidal tract, or
optogenetic silencing of the corticospinal neurons led to motor
deficits (Baird et al., 2001;Farr and Whishaw, 2002;Starkey et al.,
2005;Ueno et al., 2018). The Sulf1/2 DKO mice showed impaired
performance in the staircase test and single pellet-reaching tests,
although their gross movements and locomotion appeared to
be normal, indicating deficits in fine motor movement. In the
DKO mice, in the single pellet-reaching test, the trajectories of
the distal component of the forelimb and speed control near the
target were different from those in the wild-type mice, indicating
that the DKO mice use different goal-directed action strategies.
We examined the correlation between the parameters obtained
from the kinematic measurement in the single pellet-reaching test
(performance, trajectory length, and speed) and the anatomical
defects of the CST (signal intensity of the PKCγstaining in the
contralateral/ipsilateral and dorsal/lateral funiculi) in the DKO
mice, but we could not find any significant correlation between
them. Analysis of the synaptic connections in the spinal cord may
be necessary, although it appears to be difficult to attribute some
aspect of the behavioral deficits to simple anatomical phenotypes.
In rodents, CST axons originate from the motor,
somatosensory, parietal, cingulate, visual, and prefrontal
regions and mediate many different functions (Lemon, 2008;
Welniarz et al., 2017a). Therefore, movement impairment in
Sulf1/2 DKO mice may be caused by deficits of CST function
that include descending control of afferent inputs and gating and
gain control of the spinal reflex in addition to excitation of motor
neurons (Lemon, 2008;Welniarz et al., 2017a). Involvement
of the CST fibers originating from the somatosensory cortex
or other areas that affect their performance (Lemon, 2008;
Wang et al., 2017;Ueno et al., 2018) should also be considered.
Furthermore, it is also important to note that sensory feedback to
the CST plays a critical role in controlling movement (Seki et al.,
2003;Bui et al., 2013;Bourane et al., 2015). Although the sensory
function assessed by the hot plate test was normal in the DKO
mice, the possibility cannot be excluded that the sensory deficits
due to the abnormality of the CST fiber projections from the
sensory cortex affected the performance in the behavioral tests.
To understand the mechanism of impaired motor movements
in Sulf1/2 DKO mice, it will be important to determine whether
motor deficits are caused by reduction in the number of normal
crossed fibers or by interference in the normal functions of the
crossed fibers by the presence of abnormal ipsilateral fibers.
DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to
the corresponding author.
ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Care
and Use Committee of the University of Tsukuba.
AUTHOR CONTRIBUTIONS
SA, TO, KK-M, and MM designed the research and performed
the experiments. SA, MA, and KK-M performed the rotarod
and open field tests. TD, TK, and MM performed the EGM
recording. SA, TO, and TD analyzed the data. AT supervised
SA and evaluated the clinical implications. SA, TO, KK-M, and
MM wrote the manuscript. All the authors read and approved
the final manuscript.
FUNDING
This work was partly supported by Kakenhi grants (Grant
Numbers 22123006 and 25293065) from MEXT and the JSPS and
by research grants from the Takeda Science Foundation and the
Naito Foundation.
ACKNOWLEDGMENTS
We thank Drs. Noriyuki Higo, Hiroshi Nishimaru, Hiroshi
Kameda, Satoshi Fukuda, Naoyuki Murabe, Naohito Terui, and
Marika Kato for useful comments and Flaminia Miyamasu
for critical reading of the manuscript. We also thank Hitomi
Yuyama, Nana Kagaya, Moe Imaizumi, and Ayaka Tsukakoshi
for their support with the experiments. The apparatuses for
the grid-walking test and single pellet-reaching test were made
by the Research Facility Center for Science and Technology,
University of Tsukuba.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fnmol.2019.
00333/full#supplementary-material
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Aizawa et al. Corticospinal Dysgenesis in Sulf1/2 Mutants
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Conflict of Interest: The authors declare that the research was conducted in the
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