and fig. S18, J to Q). PlexA1fl/fl;Emx1-Cre mice also
outperformed control mice during a sticky tape
removal test (fig. S19, A and B).
To evaluate grasping, we designed and implemented a new prehension test (fig. S20) in
which animals had to use their paws to reach,
grasp, and retrieve a food pellet (Fig. 3, E to G).
PlexA1fl/fl;AAV1-Cre mice exhibited significantly
higher grasping success rates than those of controls (PlexA1fl/+;AAV1-Cre and PlexA1fl/fl;AAV1-td),
whereas pellet consumption time was indistinguishable between the two groups (Fig. 3, H to
K, and movies S5 and S6). We also found no obvious deficits in grip strength (fig. S19C) or grid-walking in PlexA1fl/fl;Emx1-Cre mice (fig. S19D).
Behavioral changes were not associated with any
obvious changes in cortico-cortical projections,
cortical lamination or dendritic development
(by means of Golgi analysis) of CS neurons in
PlexA1fl/fl;Emx1-Cre mice (fig. S21).
The presence of CM connections and the
vlCST in adult PlexA1 mutant mice bear a remarkable resemblance to human CS circuits,
prompting us to examine PLEXA1 expression
within the developing human motor cortex. Human midfetal cortices at 20 post-conceptional
weeks (pcw), which corresponds to the early postnatal mouse cortex, showed very weak PLEXA1
expression in layer 5 CS neurons, but strong
expression in layer 6 of the putative motor cortex, which does not give rise to CST projections
To determine whether species-dependent cis-
regulatory elements might define PLEXA1 ex-
pression levels in layer 5, we first identified and
compared putative orthologous enhancer re-
gions between humans and mice. Enhancers
were identified on the basis of features indicative
of active regulatory regions, including H3K4me3
and H3K27ac histone marks, deoxyribonuclease
hypersensitivity, and DNA conservation across
mammals. This resulted in a ~5-kb putative or-
thologous enhancer in humans and mice (fig.
S22). Within the putative human enhancer, we
identified a total of 28 putative FEZF2 binding
sites. FEZF2 (also known as FEZL and ZFP312),
encoding a zinc-finger transcription factor (14)
required for CS tract development (15), was
expressed in the putative layer 5 of the human
late mid-fetal neocortex (fig. S26A) or in the
putative layer 5 of the human brain at 22 pcw
(fig. S26A). Three FEZF2 binding sites corre-
spond to the typical “CTNCANCN” Fezf2 bind-
ing site (figs. S23 to S25, blue bars) (16), with the
remaining 25 resembling a recently described
“CGCCGC” element (figs. S23 to S25, green and
red bars) (17). Five of the total 28 sites were con-
served in both humans and mice (fig. S24, green
bars), whereas 23 of them were only found in
humans, resulting in a putative homotypic clus-
ter of 23 human FEZF2 binding sites (Fig. 4B
and figs. S22A and S23 to S25) (18, 19). Humans,
chimpanzees, gorillas, orangutans, and ba-
boons, which all have CM connections (2), all
possess these FEZF2 binding sites in their pu-
tative PlexA1 cis-regulatory elements (Fig. 4B
and figs. S23 to S25). In contrast, mice, rats, and
rabbits—as well as some primates that lack CM
connections, such as marmosets and bushbabies
(2)—either lack these FEZF2 binding sites com-
pletely or have nucleotide mismatches that
are predicted to decrease FEZF2 binding (Fig.
4B and figs. S23 to S25). Electrophoretic mo-
bility shift assays (EMSAs) demonstrated bind-
ing of FEZF2 to the human FEZF2 binding site
but weaker binding to the homologous mouse
sequence and human sequence with point mu-
tations (making it identical to the mouse se-
quence) (fig. S26B).
Using in vitro luciferase assays, we found that
FEZF2 represses the transcriptional activity
of the human, but not mouse, cis-regulatory
elements (Fig. 4C and fig. S26, C and D). Constructs with point mutations in both FEZF2 motifs showed a complete loss of FEZF2-mediated
repression (Fig. 4C). Enhancer analysis by trans-fecting constructs with the FEZF2 cis-regulatory
elements to primary cortical neuron cultures
from wild-type and Fezf2 mutant mice demonstrated a strong FEZF2-mediated repression
of the putative human cis-regulatory elements
in BCL11B+ (CTIP2+) layer 5 cortical neurons
(figs. S27 and S28). Further analysis of transgenic mice expressing GFP under the human
FEZF2 cis-regulatory elements revealed that
the putative human cis-regulatory elements
drove strong expression of GFP in layer 6, but
not layer 5, of the motor cortex, recapitulating the PLEXA1 expression in the human motor
cortex (Fig. 4, D and E, and fig. S29). Transgenic mice with the human FEZF2 binding
sites mutated drove robust expression in layer
5 neurons and CS axons (Fig. 4, F and G, and
fig. S29). Expression of PlexA1 was not altered
in the cortices of Fezf2 mutant mice at P0 (fig.
S26, E and F).
Considering that CM connections seem beneficial for mice, why are they eliminated? Perhaps increased manual dexterity confers no
fitness advantages to quadrupedal animals,
or perhaps it even imposes a fitness burden.
For example, maintenance of CM connections
in mice may disrupt the development and function of other spinal motor circuits, such as those
for forelimb locomotion rather than manipulation. Another question lies in the preservation
of transient CM connections in wild-type mice.
Perhaps they play a temporary developmental
role in assisting the establishment of other spinal
neural circuits. Our findings, providing insight
into the specific contributions of CM connections to dexterous manipulations by mice, serve
as a stepping stone toward answering these questions and present a potential mechanism for how
CM connections emerged during mammalian
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We are grateful to M. Baccei, J. N. Betley, K. Campbell, S. Crone,
C. Gu, T. Isa, D. Ladle, M. Nakafuku, and R. Yu for critical
comments on the manuscript. We thank Boston Children’s Hospital
Viral Core (supported by core grant NEI 5P30EY012196-17) for
making the AAV6-G virus. We thank P. Arlotta for the Fezf2
construct. Z.G. and E.B. were supported by the Graduate Summer
Undergraduate Mentoring Program at the University of Cincinnati.
M. T. W. is supported by the National Institutes of Health (NIH)
grants R01 NS099068 and R21 HG008186, Lupus Research
Alliance “Novel Approaches” award, and a CCHMC CpG Pilot
Study award. N.S. is supported by NIH grants MH106934 and
MH103339. J.H.M. and Y. Y. are supported by NIH grants NS079569
and NS093002, respectively. All data are available in the main
texts and supplementary materials. Z.G. and Y. Y. conceived of
the project and contributed to experimental design and
interpretation. Z.G. performed most of the experiments. Z.G.,
J.K., and J.H.M. designed the cortico-muscular electrophysiology
assay, performed EMG recordings, and analyzed and interpreted
the electrophysiological data. Y.I.K., W.H., Z.L., Z.L., F.L., S.P.,
X.X., and N.S. performed the in situ hybridizations using human
tissues, chromatin immunoprecipitation–sequencing using human
and mouse tissues, and culture experiments using Fezf2-floxed
mice. S. Y. contributed to cloning of the human and mouse genomic
regions and the luciferase assays. E.B. performed the grid-walking
test and contributed to the immunochemistry studies. M.U.
purified rabies viruses and assisted in the dorsal hemisection
surgery. S. W. and M.-R.R. analyzed the cortical layers and cortico-cortical projections and also conducted the Golgi analyses. J.S.
and B.G. performed the EMSA analyses. M. T. W. performed the
cross-species cis-regulatory element comparisons. A.K. provided
the Sema6D mutant mice. Y. Y. supervised all aspects of the work,
and Z.G., J.H.M, and Y. Y. prepared the manuscript with
contributions from other authors.
Materials and Methods
Figs. S1 to S29
Movies S1 to S6
4 April 2017; accepted 12 June 2017
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