Possible involvement of SINEs in mammalian-specificbrain formation
Takeshi Sasaki*, Hidenori Nishihara*, Mika Hirakawa†, Koji Fujimura*, Mikiko Tanaka*, Nobuhiro Kokubo*,
Chiharu Kimura-Yoshida‡, Isao Matsuo‡, Kenta Sumiyama§, Naruya Saitou§, Tomomi Shimogori¶, and Norihiro Okada*储
*Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-21 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501,
Japan; †Bioinformatics Center, Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan; ‡Osaka Medical Center andResearch Institute for Maternal and Child Health, Osaka Prefectural Hospital Organization, Izumi, Osaka 594-1101, Japan; §National Instituteof Genetics, Mishima, Shizuoka 411-8540, Japan; and ¶RIKEN Brain Science Institute, 2-1 Hirosawa Wako City, Saitama 351-0198, Japan
Edited by Wen-Hsiung Li, University of Chicago, Chicago, IL, and approved January 23, 2008 (received for review October 3, 2007)

Retroposons, such as short interspersed elements (SINEs) and longinterspersed elements (LINEs), are the major constituents of higher
vertebrate genomes. Although there are many examples of retroposons’ acquiring function, none has been implicated in the morphological innovations specific to a certain taxonomic group. We previously characterized a SINE family, AmnSINE1, members of whichconstitute a part of conserved noncoding elements (CNEs) in mammalian genomes. We proposed that this family acquired genomic
functionality or was exapted after retropositioning in a mammalianancestor. Here we identified 53 new AmnSINE1 loci and refined 124
total loci, two of which were further analyzed. Using a mouseenhancer assay, we demonstrate that one SINE locus, AS071, 178 kbp
from the gene FGF8 (fibroblast growth factor 8), is an enhancer that
recapitulates FGF8 expression in two regions of the developing
forebrain, namely the diencephalon and the hypothalamus. Our
gain-of-function analysis revealed that FGF8 expression in the diencephalon controls patterning of thalamic nuclei, which act as a relay
center of the neocortex, suggesting a role for FGF8 in mammalianspecific forebrain patterning. Furthermore, we demonstrated that thelocus, AS021, 392 kbp from the gene SATB2, controls gene expression
in the lateral telencephalon, which is thought to be a signaling center
during development. These results suggest important roles for SINEs
in the development of the mammalian neuronal network, a part ofwhich was initiated with the exaptation of AmnSINE1 in a common
mammalian ancestor.
conserved noncoding element 兩 enhancer 兩 evolution 兩 mouseR

etroposons, including SINEs, LINEs, and long-terminal
repeat or LTR retrotransposons, propagate within the hostgenome via RNA intermediates (1–3). Despite the abundance ofretroposons in mammalian genomes (e.g., 42% and 37% of
human and mouse genomes, respectively) (4, 5), most are
nonfunctional and are regarded as genomic parasites or ‘‘junkDNA.’’ However, in several cases, copies of retroposons havebeen involved in generating new cis-regulatory elements forprocesses such as alternative splicing, mRNA polyadenylation,and promoter activity (6), all of which are examples of exaptation(7, 8). The idea that retroposons may impact genome evolution
and functionality (8–10) has been gradually accepted. However,most examples seem to have only local effects, and no example
has been reported where an inserted retroposon plays a pivotal
role in higher-order morphological or biological innovations.
On the other hand, recent genome-scale comparative analyses
have identified conserved noncoding elements (CNEs) that aresuggested to be essential for host survival (11). CNEs constitute
3–4% of the human genome, whereas protein-coding exons constitute only ⬇1.5% (4). Recently, it was reported that 16% of
eutherian-specific CNEs are derived from transposable elements(retroposons) (12, 13), which supports the importance of retroposons in evolution (14–18). This observation on CNEs is important because retroposons arose only recently in evolution andtherefore may have the potential to acquire new functions through4220 – 4225 兩 PNAS 兩 March 18, 2008 兩 vol. 105 兩 no. 11

exaptation. We recently identified a new SINE family, AmnSINE1,
in the genomes of Amniota (mammals, birds, and reptiles). The⬇100 copies of AmnSINE1s are highly conserved as mammalianspecific CNEs (16), suggesting that these SINEs acquired a function(exapted) during the emergence of mammals (or therians) (Fig. 1).
Therefore, these AmnSINE1s are the best available mammalianspecific CNEs to study the evolutionary impact of retroposons onmorphological changes among mammals (16).
Studying brain development raises two fundamental questions(19). What characteristics of the brain make mammals distinct
from other vertebrates? How have these characteristics evolved?
In vertebrates, the anterior neural epithelium undergoes morphological subdivisions during development to generate vesiclelike structures known as the forebrain, midbrain, and hindbrain.
The forebrain further divides into the telencephalon and the
diencephalon. The telencephalon gives rise to the neocortex,
basal ganglia, and hippocampus, whereas the diencephalon
develops into the thalamus, prethalamus, and pretectum. Several
patterning centers used during development have been wellcharacterized and are generated on boundaries or ridges of eachregion, such as the anterior neural ridge, the zona limitans
intrathalamica, and the midbrain–hindbrain boundary. The patterning centers are thought to establish the regional identities bysecreting signaling molecules such as Shh, Wnts, and Fgfs (20,21). The mammalian-specific brain is identified by three characteristics (19, 22): (i) the six-layered neocortex in the dorsal
pallium (the pallial area in birds and reptiles); (ii) generation of
the Cajal–Retzius cells, one of whose sources is generated in thepallial–subpallial boundary, and migrate tangentially toward thedorsal telencephalon; and (iii) the absence of the dorsal ventricular ridge, which has been commonly observed in the brainsof birds and reptiles. The molecular mechanisms that generatethese characteristics are largely unknown. In this study wedemonstrate a potential role for SINEs in controlling gene
expression and activity during forebrain development. Because
the mammalian forebrain is evolutionarily unique, SINEs may
have played a role in mammalian-specific evolution.
Results and Discussion
Enhancer Activity of the AS071 Locus for FGF8 Expression. To eluci-

date the function of the conserved AmnSINE1 loci, we recharAuthor contributions: N.O. designed research; T. Sasaki, H.N., M.H., K.F., M.T., N.K., C.K.-Y.,
I.M., K.S., N.S., and T. Shimogori performed research; K.S. and N.S. contributed new
reagents/analytic tools; T. Sasaki, M.H., K.F., M.T., N.K., C.K.-Y., and I.M. analyzed data; andT. Shimogori and N.O. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
储To whom correspondence should be addressed. E-mail: nokada@bio.titech.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/
0709398105/DC1.
© 2008 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0709398105

Exaptation burst

opossum

Amniotamouse

Mammalia

Origin of
AmnSINE1humanbirds, reptiles
amphibians
Fig. 1. Exaptation of AmnSINE1s in a common ancestor of mammals. The
known phylogeny of tetrapods is shown with the time of exaptation of
AmnSINE1 sequences. AmnSINE1 originated in a common ancestor of amniotes, and multiple copies acquired function in a common ancestor of
mammals.

acterized conserved AmnSINE1 loci by updating the homology
search as described in Methods. We refined 124 AmnSINE1 lociand 974 neighboring genes. The 124 loci are scattered among thehuman chromosomes (except chromosomes 19, 21, 22, and Y)
but are not evenly distributed among the chromosomes (Fig. 2).
To test whether the conserved AmnSINE1 loci regulate theexpression of adjacent genes, we used a ␤-galactosidase (lacZ)transgenic mouse system. We first focused on locus AS071,
located 178 kbp downstream from FGF8 (Fig. 3a). The 570 bpof the putative full-length AmnSINE1 element (red horizontalbar) appears to be conserved among mammals (Fig. 3b). Synteny
is conserved between human and mouse in this region (data notshown). The mouse sequence of the AS071 conserved region(600 bp) was cloned upstream of a minimal heat-shock protein
68 (Hsp68) promoter coupled to the lacZ reporter gene [SI Fig.
7 in supporting information (SI) Appendix], and this was injected
into a fertilized mouse oocyte. To understand spatial andtemporal lacZ expression in the mouse embryo, whole-embryo
staining for lacZ activity was performed at embryonic day 10.5
(E10.5), E11.5, and E13.5 (Fig. 4 a–h and SI Table 1 in SI

Fig. 3. Characterization of the AS071 locus. (a) The 2-Mb region containing
three AmnSINE1 elements in human chromosome 10. Coordinate (Top),AmnSINE1-inserted loci (Middle), and gene annotations (Bottom) are basedon the hg18 assembly in the UCSC genome browser (http://genome.ucsc.edu).
(b) The 3.0-kb region around the AS071 locus and conservation among vertebrate species. The 570-bp region of the putative full-length AmnSINE1
element (red horizontal bar) appears to be conserved among mammals,
including opossum. Within the bar, only 200 bp (red fill) can be aligned with
the AmnSINE1 consensus sequence. The sequences indicated by the open bar
regions of the SINE have decayed and thus cannot be precisely aligned. The
arrow indicates the direction of the element (5⬘ to 3⬘).

Appendix). Consistent expression of LacZ is observed in independent transgenic embryos in the lateral wall of the diencephalon, dorsal midline of the caudal telencephalon, and the hypothalamus (Fig. 4 a–c and SI Fig. 8 in SI Appendix) at E10.5 (30embryos of 50), E11.5 (eight of 18), and E13.5 (eight of 17).
FGF8 is well established as a signaling molecule, and its expression pattern and function during CNS development in mice andchicks have been reported (23–28). Because the expression of
AS071 is restricted to the lateral diencephalon and hypothalamus, we compared the expression pattern of AS071 lacZ withthat of Fgf8 by in situ hybridization at E10.5 and E11.5 (Fig. 4 i,j, and l). Interestingly, lacZ expression and Fgf8 expression in thelateral diencephalon (red arrows) as well as in the hypothalamus(white arrowheads) coincided exactly (Fig. 4 a, b, d, i, j, and l).
Sagittal (Fig. 4 c and k) and transverse (Fig. 4 e–h and m–p)sections of brain showed detailed expression of lacZ and Fgf8,
revealing identical expression in the diencephalon (red arrows)
and hypothalamus (white arrowheads). These data suggest thepossible role of AS071 as a specific enhancer of Fgf8 in the
diencephalon and hypothalamus of the developing forebrain.

Fig. 2. Positions of the 124 conserved AmnSINE1 loci on the human chromosomes. Each locus is indicated by an arrow.

Sasaki et al.

function of FGF8 in diencephalon, we used in utero electroporation (24) to overexpress FGF8 in the developing diencephalon
at E10.5 (Fig. 5a). With the possibility that FGF8 could be
involved in area patterning of the diencephalon through theanterior/posterior axis, we tested the pattern of thalamic nuclei
at postnatal day 6 (P6) by cytochrome oxidase staining and Nissl
staining (Fig. 5 b–d). The somatosensory nucleus in the thalamus, which represents the pattern of whiskers on the animal’ssnout (barreloid), was easily distinguished in both sides of thethalamus in the control animal (Fig. 5b). Unilateral FGF8electroporation of the lateral diencephalon caused one side of
the barreloid to shift and shrink as compared with the control(Fig. 5c, bracket). Nissl staining revealed a shifted mammillothalamic tract, which runs through the boundary of the thalamus
and prethalamus. Conversely, the habenulopeduncular tract,which runs through the pretectum and thalamus, was not altered,
PNAS 兩 March 18, 2008 兩 vol. 105 兩 no. 11 兩 4221

EVOLUTION

Function of FGF8 Expression in Diencephalon. To elucidate theFig. 4. Comparison of the lacZ expression pattern in an AS071 transgenic mouse (a–h) with the FGF8 mRNA expression pattern in mouse (i–p) or chick (q andr) embryos. Lateral (a, i, and q) and dorsal (b, j, and r) views of embryos are shown. For hybridization and staining of the head, surface ectoderm and mesenchyme
were removed. (c and k) Sagittal section of the head region. (d and l) Lateral view of the head. Transverse sections (e, g, m, and o) and corresponding close-up
views ( f, h, n, and p) are shown. lacZ and FGF8 mRNA expression in the lateral diencephalon and hypothalamus are indicated by red arrows, and those in theventral diencephalon are indicated by a white arrowhead. FGF8 mRNA expression at the midbrain– hindbrain boundary and the commissural plate are indicatedby black and green arrowheads, respectively. The dashed lines indicates where the embryos were sectioned for the staining shown in e, g, m, and o. [Scale bars:
500 ␮m (a–e, g, i–m, q, and r), 100 ␮m ( f, h, n, and o), and 50 ␮m (p).]suggesting that repatterning upon FGF8 overexpression occurred specific to the thalamus but not to the pretectum (Fig. 5d).
During CNS development, the diencephalon dives down in
between the telencephalon, and its bent longitudinal axis causes
its true anterior/posterior axis to rotate 90° (T. Shimogori,unpublished data). Accordingly, the shift of the barreloid upon
FGF8 expression is toward the posterior, which is linking to therole of this protein in other parts of the CNS (24, 27). These data
demonstrate that the expression of FGF8 in the diencephalon
controls patterning of thalamic nuclei.
FGF8 Expression in Diencephalon Is Mammalian-Specific. Because

conservation of the AS071 locus is observed only in mammals,
the enhancer function of this locus is expected to be mammalianspecific. Based on our results that AS071 is likely to enhance
FGF8 expression, we investigated FGF8 expression in chick andperformed in situ hybridization for Fgf8 in Hamburger–
4222 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0709398105

Hamilton (HH) stage-23 chick brain to compare with the mouse
results (Fig. 4 q and r). Although the chick diencephalon showed
expression of Fgf8, it was weaker than that of the mouse(compare Fig. 4 i and j with q and r, red arrows), whereas strong
expression in the anterior telencephalon (green arrowheads) was
observed in both mouse and chick embryos (Fig. 4 i, j, q, and r).
This result suggests the existence of mammal-specific enhanced
expression of FGF8 in diencephalon, which organizes mammalian thalamic patterning differently from other vertebrates.
Although overexpression of FGF8 in chick embryos changes
regional identity (29), the different organization of thalamic
nuclei between chicks and mice makes direct comparison difficult. However, our findings suggest local gain of function of
specific protein by SINE insertion, possibly changing the functional area identity in specific region of the brain. This patterningchange may also have the chance to alter the pattern of its wired
region of the brain.
Sasaki et al.

The functional significance of the dorsal forebrain, where
FGF8 is expressed, has not been well examined. Based on our
results, we propose a role for FGF8 in thalamic pattern formation, and such function may have been introduced by enhancer
activity of the AS071 locus. In addition, we confirmed theexpression of signaling molecules such as Bmp4, noggin, and Wnt
in the dorsal forebrain and observed that Otx2 (30) expression
coincided with that of Fgf8 (SI Fig. 9 in SI Appendix), supporting
the assertion that this region constitutes a patterning center forthe diencephalon. How the regional identity of the thalamus andthalamic patterning are independently achieved in the diencephalon using signals from this center remains unclear.
Enhancer Activity of the AS021 Locus. Given the above results, we

examined other loci, looking for enhancer activities among the
124 conserved AmnSINE1 loci. We found that locus AS021 inhuman chromosome 2 (Fig. 6a) contains a 600-bp region of
AmnSINE1 highly conserved among mammals (Fig. 6b). To test
its function as an enhancer of gene expression, we used the same
method as above. We assayed the enhancer activity in AS021
locus at E10.5, E11.5, E12.5, and E13.5 (SI Table 1 in SI
Appendix). At E10.5 we could not observe any consistent lacZ
expression patterns. The first consistent lacZ expressions wereobserved in two of 10 embryos in the dorsolateral side oftelencephalon at E11.5 (SI Fig. 10a in SI Appendix, arrowhead).
At E12.5 the expression patterns in the dorsolateral side oftelencephalon became more noticeable (SI Fig. 10 b and c in SI
Appendix). In the later stage, at E13.5, 13 of 23 embryos consistently showed lacZ expression in the neocortex of telencephalon(Fig. 6 c–j and SI Fig. 10 d–h in SI Appendix). Judging from thedifferences of limb development of embryos, we can discriminate
embryos at relatively early phase (Fig. 6 c–f ) from those at latephase (Fig. 6 g–j) even at E13.5. It appears that the lacZ expression
patterns in the telencephalon expanded from the restricted area oftelencephalon (dorsolateral side, Fig. 6 c–f ) to the entire of
neocortex (Fig. 6 g–j) as the developmental stage progressed. We
found that locus AS021 is located 390 kbp upstream from the gene
SATB2 (Fig. 6a), which was recently shown to be a multifunctional
determinant of craniofacial patterning and osteoblast differentiation (31). SATB2 is also expressed in the telencephalon at E13.5(32), suggesting a role in the developing telencephalon. Because
Sasaki et al.

SATB2 is multifunctional and is expressed ubiquitously, including in
the brain and skeleton, it is difficult to prove that locus AS021
enhances neocortical subexpression of SATB2 based on a lacZ
expression pattern. Circumstantial evidence, however, suggests thatit does. First, SATB2 is the only gene involved in development in theAS021-neighboring region we searched (2 Mb in total), which
includes 10 mouse genes (SI Table 2 in SI Appendix). Second, LacZ
staining in the neocortex was first observed at E11.5–E12.5. (SITable 1 in SI Appendix). This developmental expression pattern is
consistent with that reported for SATB2 (31) (SI Fig. 10 in SI
Appendix). Third, a brain section study showed that lacZ staining
was first observed in the cortical plate at E13.5 (Fig. 6 c–f ) and then
expanded into other neocortex areas (the intermediate zone; Fig.
6 g–j), a developmental expression pattern consistent with that of
SATB2 (32) (SI Fig. 10 in SI Appendix). Furthermore, detailed
expression of lacZ was evident in sectioned brain tissue, which
revealed restricted expression in the lateral telencephalon (Fig. 6 e
and f ).
The cerebral cortex has a laminar organization, which showsa birth date-dependent migration manner (i.e., early-born/inside,late-born/outside). Correct ordering of lamina formation requires Reelin expression and several sources of Reelinexpressing Cajal–Retzius cells (33). One of the sources for a
subpopulation of Reelin-positive Cajal–Retzius cells is from theborder of the pallium and subpallium (33, 34) (antihem), where
cells stained by lacZ (arrows in Fig. 6f ) appeared to be first
generated. Cells generated from the antihem, as labeled by
Dbx1-LacZ expression, migrate tangentially toward the dorsal
telencephalon, which seems to be lacking in the chick telencephalon (ref. 33 and A. Pierani, personal communication). This
result strongly suggests a mammal-specific contribution of locus
AS021 activity to the development of a mammal-specific fundamental structure (19). However, further study is required tograsp a total understanding of this phenomenon.
SINEs as Distal Enhancers. To date, we have examined 10 AmnSINE1

loci and demonstrated that the two loci, AS071 and AS021, function
as distal transcriptional enhancers in developing mouse embryos. It
should be noted that both enhancers are specific to the developing
forebrain and have functions that are mammalian-specific. Although we speculate that these enhancers contribute to mammaPNAS 兩 March 18, 2008 兩 vol. 105 兩 no. 11 兩 4223

EVOLUTION

Fig. 5. Gain-of-function analysis of FGF8 in developing diencephalons. (a) Scheme for in utero electroporation. Plasmid solution was injected in the thirdventricle of E10.5 embryos in utero, followed by insertion of needle-type electrodes. A series of three square-wave current pulses (7 V, 100 ms, three times) was
delivered, resulting in gene transfection into a restricted region in the unilateral diencephalons (the region is indicated by ⫹, and ⫺ indicates the region where
gene transfection was not performed). Gene transfer was visualized by GFP after 48 h of electroporation, demonstrating localized expression of the transgene.
(b–d) Coronal section of P6 brains processed for cytochrome oxidase (CO) histochemistry (b and c) and Nissl staining (d). The control brain shows the symmetricposition of the barreloid (b), whereas the FGF8-electroporated brain shows a posteriorly shifted and shrunken barreloid on the electroporated side (c) as wellas mammillothalamic tract (MMT). Nissl staining reveals the habenulopeduncular tract (HPT), which is not altered by FGF8 electroporation (d).

Bejerano’s group (36) recently discovered ⬎10,000 transposons
conserved among mammals that are located near developmental
genes in the human genome. Accordingly, we speculate that theenhancers in loci AS071 and AS021 play important roles in brain
development as a part of the neuronal networks generated uponinsertion of AmnSINE1s in a common mammalian ancestor, which
led to the unique evolution of the mammalian brain.
The fact that many CNEs are derived from transposable
elements [e.g., 16% in Eutheria (12)] leads to the speculationthat other SINEs in various organisms may also play importantbiological roles such as enhancers. AmnSINE1 belongs to the
Deu-SINE superfamily, having a conserved domain, the Deu
domain, in the middle of the SINE. Members of this group
include coelacanth LmeSINE1, zebrafish SINE3, and amphioxus
BflSINE1 (16). Because they share this conserved domain, we
speculate that these SINEs may have important functions.
Furthermore, other superfamilies of SINEs, such as V-SINEs(37) and CORE-SINEs (38, 39), have been reported. These
SINEs also have a highly conserved domain in the middle of theirsequences, even though the overall sequences are unique. We
anticipate that functions for these SINEs with the conserved
domain will be elucidated in the near future (40).
Methods
Screening the AmnSINE1 Loci. We searched AmnSINE1 sequences in the human[University of California, Santa Cruz (UCSC) hg18, National Center for Biotechnology Information Build 36.1] and mouse (UCSC mm8, National Center for
Biotechnology InformationBuild 36) genomes using the FASTA program. SINE
conservation was evaluated based on the phastCons Conserved Elements andthe phastCons scores available in the UCSC database (http://genome.
ucsc.edu). We searched the refFlat data in the UCSC database for genes having
a transcription start site within 1 Mbp (for both strands) from the midpoint of
a conserved AmnSINE1 sequence.
Mouse Transgenic Enhancer Assay. The AS071 and AS021 loci were amplified
from MCH mouse DNA by PCR, and each amplified fragment was subcloned
into the HSF51 vector containing the mouse hsp68 basic promoter and thebacterial LacZ coding region (41). The subcloned construct (10 ng/␮l), digested
with ScaI, was purified and injected into pronuclei as described (41). Fertilized
eggs were collected from pregnant B6C3F1 females. Transient transgenic
embryos were harvested at E10.5–E13.5 and were stained to visualize ␤-galactosidase activity (41).
Fig. 6. Characterization of the AS021 locus. (a) The 2-Mb region containing an
AmnSINE1 element in the AS021 locus in human chromosome 2. Coordinate(Top), AS021 locus (Middle), and gene annotations (Bottom) are based on the
hg18 assembly in the UCSC genome browser (http://genome.ucsc.edu). (b) The
3.0-kb region around the AS021 locus and conservation among vertebrate species. The red horizontal bar indicates the putative full-length AmnSINE1 element.
Within the bar, the red fill indicates an element that can be aligned with the
AmnSINE1 consensus sequence. The display form is in accord with Fig. 3b. (c–j) lacZ
expression patterns in an AS021 transgenic mouse embryos (E13.5). Lateral (c andg) and frontal (d and h) views of lacZ-stained whole-mount embryo are shown.
The dashed line indicates where the embryo was sectioned for the staining shownin the coronal section (e and i). Arrowheads indicate lacZ expression in the
neocortex. Arrows indicate the antihem (the border of the pallium and subpallium). [Scale bars: 2.0 mm (c, d, g, and h), 0.5 mm (e and i), and 0.2 mm ( f and j).]lian-specific brain formation, it is not certain whether insertions ofthese SINEs first triggered formation of the unique mammalianbrain in a common ancestor of mammals, because the redundancyof enhancers is frequently observed. From the conservation of theenhancer sequence at the AS071 or AS021 locus among mammals,
however, we conclude that they contributed to mammalian-specificbrain formation through natural selection regardless of the possibility that future knockout experiments may not produce phenotypic alterations (35).
When we assessed the distribution of all 124 AmnSINE1 loci, we
discovered that one-fourth (32 of 124 loci) are located near genes
involved in brain development. It should also be mentioned that4224 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0709398105

Genotyping Analysis. We extracted total genomic DNA from yolk sacs of alltransient transgenic embryos. Positive transgenic animals were confirmed by PCR.

In Situ Hybridization. The surface ectoderm of E10.5 and E11.5 mouse embryos
and HH23 chick embryos were removed from the head region with forceps indiethylpyrocarbonate (DEPC)-treated PBS. The embryos were fixed overnightin 4% paraformaldehyde dissolved in DEPC-treated PBS at 4°C. Whole-mountin situ hybridization in mouse and chick embryos was performed as described(42). After in situ hybridization, mouse embryos were processed for paraffin
sectioning by 8-␮m sections.
In Utero Electroporation. Electroporation was performed as described (24)with modifications to electroporate the diencephalon. DNA solution was
injected into the third ventricle, followed by negative electrode insertion into
the lumen of the third ventricle. The positive electrode was placed outside,
near the head of the embryo. A series of three square-wave current pulses (7V, 100 ms) was delivered, resulting in gene transfection into one side of the
diencephalic wall.
Additional Details. Full methods and associated references are available in SI
Appendix.
ACKNOWLEDGMENTS. We thank Dr. Gail Martin (University of California, San
Francisco) for the Fgf8 in situ probe and Dr. Yumiko Saga for help in pursuingthis work by using the facility of the National Institute of Genetics. This work
was supported by research grants from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (to N.O.) and from the National
Institute of Genetics Cooperative Research Program (Grants 2006-A75 and2007-B3 to N.O.).

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