DIVISION OF BRAIN BIOLOGYProfessor

Associate Professor

YAMAMORI, Tetsuo

WATAKABE, Akiya

Assistant Professor:

KOMINE, Yuriko
SADAKANE, Osamu
Assistant Professor (Specially appointed):
KOMATSU, Yusuke#NIBB Research Fellow:
OHTSUKA, MasanariTechnical Staff:
OHSAWA, Sonoko
Postdoctoral Fellow:
TAKAJI, Masafumi#
OHTSUKA, Masanari*
HATA, Katsusuke#
SASAKI, Tetsuya
Visiting Scientist:
HIRAKAWA, Reiko
Graduate Student:
SHUKLA, RammohanTechnical Assistant:
NAKAMURA, Tohru
TAKEDA, Yuta#NAKAGAMI, Yuki
MORITA, Junko
IMAI, Akiko
KOTANI, Keiko
KON, Yayoi
IMOTO, EikoHIRAYAMA, Yuka#
KAJITANI, Tomoki#
TAKAHASHI, Yoichi#

Figure 1. Cover letter for J.
Comp. Neurology, Volume520, Issue 16.

#: SRPBS (Strategic Research Program for Brain Sciences), NIPS

We continue to study genes that are expressed in specificareas of the neocortex in order to understand the principles
governing the formation of the primate brain. In addition,
this year we have reported a novel genetic manipulation
method to visualize cortical projection neurons withretrograde TET-off lentiviral vector, area-specificsubstratification of deep layer neurons in the rat cortex, andthe behavioral analysis of zfhx2-deficient mice as follows.

I. Area-specific substratification of deep layer
neurons in the rat cortex
To identify cell types for fine mapping of neuronal circuits,
we studied area-specific sublamina structures of the rat
cerebral cortex using cholecystokinin (cck) and purkinje cell
protein4 (pcp4) mRNAs as the markers for the subtypes of
excitatory neurons in layers 5 and 6. We found a segregatedexpression, especially pronounced in layer 6, wherecorticothalamic and corticocortical projecting neurons reside(Figure 2). To examine the relationship between gene
expression and projection target, we injected retrograde
tracers into several thalamic subnuclei, ventral posterior(VP), posterior (PO), mediodorsal (MD), medial and lateralgeniculate nuclei (MGN and LGN); as well as into two
cortical areas (M1 and V1). This combination of tracer-in
situ hybridization (ISH) experiments revealed thatcorticocortical neurons predominantly express cck andcorticothalamic neurons predominantly express pcp4
mRNAs in all areas tested. In general, cck(+) and pcp4(+)
cells occupied the upper and lower compartment of layer 6a,respectively. However, the sublaminar distribution and theFigure 2. Expression patterns of cck and pcp4 mRNAs in various corticalareas. A: Low magnified views of the double-ISH of cck (red) and pcp4(green) mRNAs in the rat brain. PL, prelimbic cortex; Cla, claustrum;
M1, primary motor cortex; S1Tr, primary somatosensory cortex, trunk
region; S1BF, primary somatosensory cortex, barrel field; S2, secondary
somatosensory cortex; V1, primary visual cortex; V2L, secondary visualcortex lateral area. B–G: The magnified cortical areas labeled in panel A.
Scale bars = 1 mm in A; 200 μm in B–G. (Watakabe et al., J Comp
Neurol. 520, 3553-3573, 2012.)

relative abundance of cck(+) and pcp4(+) cells were quitedistinctive across areas. For example, layer 6 of the prelimbic
cortex was almost devoid of cck(+) neurons, and was
occupied instead by corticothalamic pcp4(+) neurons. In thelateral areas, such as S2, there was an additional layer ofcck(+) cells positioned below the pcp4(+) compartment. Theclaustrum, which has a tight relationship with the cortex,
mostly consisted of cck(+)/pcp4(−) cells. The combination ofgene markers and retrograde tracers revealed a distinct
sublaminar organization, with conspicuous cross-areavariation in the arrangement and relative density ofcorticothalamic connections. (Watakabe et al., J Comp
Neurol. 520, 3553-3573, 2012.)

II. Visualization of cortical projection neurons
with retrograde TET-off lentiviral vector.
We are interested in identifying and characterizing various
projection neurons that constitute the neocortical circuit.
For this purpose, we developed a novel lentiviral vector
that carries the tetracycline transactivator (tTA) and theNote: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

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National Institute for Basic Biology

Neurobiology

transgene under the TET Responsive Element promoter(TRE) on a single backbone. By pseudotyping such a vector
with modified rabies G-protein, we were able to expresspalmitoylated-GFP (palGFP) or turboFP635 (RFP) in thecorticothalamic, corticocortical, and corticopontine neuronsof mice. The high-level expression of the transgene achievedby the TET-Off system enabled us to observe characteristic
elaboration of neuronal processes for each cell type (Figure3). At higher magnification, we were able to observe finestructures such as boutons and spines as well. We also
injected our retrograde TET-Off vector to the marmoset
cortex, and therefore proved that this method can be used tolabel the long- long-distance cortical connectivity of themillimeter scale. In conclusion, our novel retrograde tracer
provides an attractive option to investigate the morphologies
of identified cortical projection neurons of various species(e.g., Figure 4) (Watakabe et al., PLoS One. 7, e46157,2012).
Figure 4. Reciprocal connectivity of marmoset V1 and V2 visualized by
StTTrR/FuG-B vector.
(A) Dark field image overlaid with RFP fluorescence indicates theinjection site. The V1/V2 border (shown by the arrowhead) was clearlydelineated by the presence of the striate of Gennari (g). The arrow
indicates the injection site. (B and C) The RFP signals around theinjection site (red) are shown with NeuN counterstaining (green). The
arrow indicates the plexus of V1 terminals in layer 4. The boxed region
contains the retrogradely labeled V2 neurons with feedback projection toV1, which is magnified in panel F. The asterisk indicates the core ofinjection, where NeuN expression is lost by local damage. Only the RFP
signals are shown in panel C. (D and E) RFP signals (red) and NeuN
stains (green) in the injection center. The contrast for the RFP signals isadjusted so that the cell bodies of the infected neurons can be delineated.
Note that the lamina positions of the infected cells are restricted mostly tolayers 2, 3 and 6. (F–H) Confocal images of RFP in V2. Panel F shows
the cell bodies and dendrites of V2 neurons that project back to V1. (G)The spines of the basal dendrites originating from one of these cells. (H)The plexus of axon terminals concentrated in layer 4 of V2 (shown by thearrow). Bar: 2 mm for A; 500 μm for B and C; 200 μm for D and E; 50μm for F and H; 10 μm for G.
Figures 3 and 4 from Watakabe et al., PLoS One. 7, e46157, 2012.

III. Behavioral analysis of zfhx2-deficient mice.

Figure 3. Retrograde infection of the corticothalamic cells by StTTrG/FuG-B vector. StTTrG/FuG-B vector was injected into the mousethalamus. (A–D) After one week (for panels A and B) or four weeks (forpanels C–G), the mouse was perfusion fixed and sliced at 40 μmthickness. The boxed areas in panels A and C were magnified in panels Band D, to show infection of corticothalamic neurons in layer 6 (indicatedby arrowheads). The injection site is indicated by the asterisk (*). Theseimages show the original green fluorescence of the infected cells. (E–G)
The immunoperoxidase detection of the GFP-expressing cells. Bar: 500μm (A and C) , 200 μm (for others). Watakabe et al., PLoS One. 7,e46157. The caudal block of the marmoset brain was parasagittally
sectioned to visualize the reciprocal connectivity between V1 and V2.

40Zfhx2 (also known as zfh-5) encodes a transcription factor
containing three homeobox domains and 18 Zn-finger
motifs. We have reported that Zfhx2 mRNA is expressed
mainly in differentiating neurons in the mouse brain and its
expression level is negatively regulated by the antisensetranscripts of Zfhx2 (Figure 5). Although the expression
profile of Zfhx2 suggests that ZFHX2 might have a role in aparticular step of neuronal differentiation, the specificfunction of the gene has not been determined. We therefore
generated a Zfhx2-deficient mouse line and performed acomprehensive battery of behavioral tests to elucidate thefunction of ZFHX2. Homozygous Zfhx2-deficient mice
showed several behavioral abnormalities, namely,
hyperactivity, enhanced depression-like behaviors, and an
aberrantly altered anxiety-like phenotype (e.g., Figure 6).
These behavioral phenotypes suggest that ZFHX2 might playroles in controlling emotional aspects through the function of
monoaminergic neurons where ZFHX2 is expressed.

Moreover, considering their phenotypes, the Zfhx2-deficient
mice may provide a novel model of human psychiatricdisorders. (Komine et al., PLoS One. 7, e53114, 2012)

Figure 5. Structure and expression of mouse Zfhx2.(A) Structure of
ZFHX2 and related proteins. ZFHX2 is a protein of 2562 amino acids
containing 18 zinc fingers (green ovals) and three homeodomains (redsquares). (B) Zfhx2, Zfhx3, and Zfhx4 mRNA detected by semiquantitative RT-PCR in various RNA sources. Note that cDNAs wereamplified for 30 cycles for brains of different developmental stages,
whereas cDNAs were amplified for 35 cycles for various adult tissues.
(C–E) Expression of Zfhx2 (C), Zfhx3 (D), and Zfhx4 (E) mRNA in theparasagittal sections of an E15.5 mouse brain. These three genes wereexpressed in substantially similar patterns with the highest expression
level of Zfhx3. (F, G) Expression of Zfhx2 mRNA (F) and the ZFHX2protein (G) was compared on adjacent coronal sections of an E15.5 brain.
mRNA expressed in the thalamic region (Th) was translated, whereas
mRNA expressed in the cerebral cortex (Cx) was not translated: thissituation made the expression patterns of ZFHX2 and ZFHX3 more alikein protein level than in mRNA level. (H–J) Expression of Zfhx2 (H),Zfhx3 (I), and Zfhx4 (J) mRNA in the coronal sections of an adult brain.
Cerebral cortex (Cx), hippocampus (Hp), thalamus (Th), caudate
putamen (CP). Expression levels of all three genes were decreased
compared with those in the embryonic brain, but Zfhx2 maintained ahigher level of expression than the others. (K–P) Expression of ZFHX2protein in the adult brain. The pyramidal layer of the hippocampus (K,Py), the suprachiasmatic nucleus (L, SCN), laterodorsal thalamic nucleus(M, LD), lateral geniculate nucleus (N, LGN), substantia nigra pars
compacta (O, SNc), and magnocellular part of the red nucleus
suprachiasmatic (P, RMC). (Q–Y) Double-color in situ hybridization with
Zfhx2(red) and tyrosine hydroxylase (Th) probes (green).

Figure 6. Locomotor activity measured in open field test. Distancetraveled (A, B),stereotypic behavior (C), vertical activity (D), and timespent in the center area (E). Measurements are blocked in 5 min (A, C, D,and E) or in 1 min (B, shown only for the first 10 min). (Komine et al.,PLoS One. 7, e53114, 2012)

Publication List
〔Original papers〕
M., Matsui, R., Kato, S., Hasegawa, T., Kasahara, H., Isa, K.,• Kinoshita,Watakabe, A., Yamamori, T., Nishimura, Y., Alstermark, B., Watanabe,••••D., Kobayashi, K., and Isa, T. (2012). Genetic dissection of the circuitfor hand dexterity in primates. Nature 487, 235-238.
Komine, Y., Takao, K., Miyakawa, T., and Yamamori, T. (2012).
Behavioral abnormalities observed in zfhx2-deficient mice. PLoS ONE7, e53114.
Takahata, T., Shukla, R., Yamamori, T., and Kaas, J.H. (2012).
Differential expression patterns of striate cortex-enriched genes among
Old World, New World, and prosimian primates. Cereb. Cortex 22,2313-2321.
Watakabe, A., Hirokawa, J., Ichinohe, N., Ohsawa, S., Kaneko, T.,
Rockland, K.S., and Yamamori, T. (2012). Area-specific substratificationof deep layer neurons in the rat cortex. J. Comp. Neurol. 520, 35533573.
Watakabe, A., Kato, S., Kobayashi, K., Takaji, M., Nakagami, Y.,
Sadakane, O., Ohtsuka, M., Hioki, H., Kaneko, T., Okuno, H.,
Kawashima, T., Bito, H., Kitamura, Y., and Yamamori, T. (2012).
Visualization of cortical projection neurons with retrograde TET-off
lentiviral vector. PLoS ONE 7, e46157.

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