RESEARCH ARTICLE 1665

Development 136, 1665-1674 (2009) doi:10.1242/dev.032714

A cluster of three long-range enhancers directs regional Shh
expression in the epithelial linings
Tomoko Sagai1, Takanori Amano1, Masaru Tamura1, Yoichi Mizushina1, Kenta Sumiyama2
and Toshihiko Shiroishi1,*
The sonic hedgehog (Shh) pathway plays indispensable roles in the morphogenesis of mouse epithelial linings of the oral cavity andrespiratory and digestive tubes. However, no enhancers that regulate regional Shh expression within the epithelial linings havebeen identified so far. In this study, comparison of genomic sequences across mammalian species and teleost fishes revealed threenovel conserved non-coding sequences (CNCSs) that cluster in a region 600 to 900 kb upstream of the transcriptional start site of themouse Shh gene. These CNCSs drive regional transgenic lacZ reporter expression in the epithelial lining of the oral cavity, pharynx,
lung and gut. Together, these enhancers recapitulate the endogenous Shh expression domain within the major epithelial linings.
Notably, genomic arrangement of the three CNCSs shows co-linearity that mirrors the order of the epithelial expression domains
along the anteroposterior body axis. The results suggest that the three CNCSs are epithelial lining-specific long-range Shhenhancers, and that their actions partition the continuous epithelial linings into three domains: ectoderm-derived oral cavity,
endoderm-derived pharynx, and respiratory and digestive tubes of the mouse. Targeted deletion of the pharyngeal epithelium
specific CNCS results in loss of endogenous Shh expression in the pharynx and postnatal lethality owing to hypoplasia of the soft
palate, epiglottis and arytenoid. Thus, this long-range enhancer is indispensable for morphogenesis of the pharyngeal apparatus.

INTRODUCTION
The Shh pathway plays a key role in multiple aspects ofmorphogenesis and development in vertebrate embryos (Chiang etal., 1996; Ingham and McMahon, 2001; Varjosalo and Taipale,2008), including dorsoventral patterning of the central nervous
system (CNS) (Echelard et al., 1993; Roelink et al., 1994) andanterorposterior patterning of the limb (Riddle et al., 1993). It also
plays an important role in the morphogenesis in the epithelial flat
sheet from the oral cavity to the hindgut, where a vast array ofmature morphologies are exhibited (Chuong et al., 2000). Targeted
deletion of Shh has shown a role for Shh signaling in thedevelopment of feathers (Yu et al., 2002), hair (St-Jacques et al.,1998), teeth (Dassule et al., 2000), lingual papillae (Hall et al.,2003), pharyngeal arches and pouches (Moore-Scott and Manley,2005), foregut (Litingtung et al., 1998), and urogenital tracts(Haraguchi et al., 2001). However, the role of the Shh pathway inlater stages of epithelial morphogenesis is poorly understood,
because these tissues are severely malformed in Shh knockout (KO)mice.
A key step towards understanding the regulatory networks that
control gene expression during morphogenesis and organogenesis
is the identification of cis-regulatory elements of developmentally
important genes. With the increasing availability of vertebrate
genomic sequences, comparison of sequences across evolutionarily
distant species has permitted the identification of numerous
conserved non-coding sequences (CNCSs) (Abbasi et al., 2007;
Boffelli et al., 2004; Frazer et al., 2004; Ghanem et al., 2003; Goode1

Mammalian Genetics Laboratory, National Institute of Genetics, Yata-1111 Mishima
Shizuoka-ken 411-8540, Japan. 2Population Genetics Laboratory, National Instituteof Genetics, Yata-1111 Mishima Shizuoka-ken 411-8540, Japan.
*Author for correspondence (e-mail: tshirois@lab.nig.ac.jp)
Accepted 10 March 2009

et al., 2005; Santagati et al., 2003; Woolfe and Elgar, 2007; Woolfe
et al., 2007; Woolfe et al., 2005). Functional analysis of theseelements has been carried out using BAC reporter transgenesis inmice (Gong et al., 2002; Jeong et al., 2006) and GFP reporter assays
in zebrafish (Goode et al., 2005; McEwen et al., 2006; Woolfe et al.,2005).
We have previously reported that deletion of a CNCS located 840
kb upstream of the transcriptional start site of Shh results in a marked
phenotype (Sagai et al., 2005). This CNCS is conserved among all
tetrapods species examined, as well as in teleost fishes (Lettice et al.,
2003; Sagai et al., 2004). Although the CNCS KO mice are viable,
endogenous limb bud expression of Shh is completely lost, resulting
in severe distal limb truncation indistinguishable from that observedin the KO mutant of the Shh-coding sequence (Chiang et al., 1996).
Subsequent cis-trans tests verified that the CNCS contains a limbspecific Shh enhancer (Lettice et al., 2002; Sagai et al., 2004).
The 1 Mb genomic region spanning from the Shh coding region
to the upstream limb-specific enhancer is unique in its low genedensity and shows exceptionally long-range synteny between
mammals and teleost fishes, with a number of CNCSs lining up in
the same order and orientation in different species (Goode et al.,2005; Woolfe et al., 2005). Comparative sequence analysis andtransgenic mouse reporter assays have uncovered three forebrain
enhancers located 300 to 450 kb upstream of the Shh-coding
sequence (Jeong and Epstein, 2003; Jeong et al., 2006).
Displacement of these regulatory elements from the Shh promoter
by chromosomal translocation is a likely cause ofholoprosencephaly (HPE) in humans (Roessler et al., 1997),
underscoring the importance of long-range enhancer elements in key
development processes. Together with floor-plate enhancers near thetranscriptional start site, these enhancers recapitulate Shh expression
in the mouse embryonic central nervous system (Epstein et al., 1999;
Jeong and Epstein, 2003; Jeong et al., 2006; Jeong et al., 2008).
However, to date, epithelial linings-specific Shh enhancers have notbeen reported.

DEVELOPMENT

KEY WORDS: Sonic hedgehog, Conserved non-coding sequence, Long-range enhancer, Epithelial lining, Mouse1666 RESEARCH ARTICLE

Here, we explore new CNCSs in the mouse 300 kb genomic region
600 to 900 kb upstream of the Shh-coding sequence by comparing themouse genome with the genomes of other mammalian species andteleost fishes. We identify a cluster of three CNCSs that drive lacZ
reporter expression in the epithelia of the oral apparatus, thepharyngeal apparatus, and the lung and gut. Interestingly, the co-linear
genomic arrangement of the three CNCSs mirrors the anteroposteriororder of their expression domains, partitioning the continuous
epithelial lining into three Shh expression domains: the ectodermderived oral cavity, the anterior endoderm-derived pharynx, andposterior endoderm-derived respiratory and digestive tubes. We also
generate KO mouse mutants that lack the CNCS that drives
expression in the pharyngeal apparatus. In these animals, endogenous
Shh expression is lost specifically in the pharyngeal epithelia, resulting
in postnatal lethality owing to hypoplasia of the soft palate, epiglottis
and arytenoid that are essential for respiratory and swallowing
functions. These results demonstrate that the Shh pathway is essential
for the morphogenesis and the development of the pharyngeal region
in the mouse.
MATERIALS AND METHODS
Animals

The Shh-coding sequence KO mouse (Shh–/–) was kindly provided by Dr
P. Beachy, and is maintained at the National Institute of Genetics (NIG),
Mishima, Japan. C57BL/6J and (C57BL/6⫻DBA/2)F1 mice werepurchased from Japan Crea (Tokyo, Japan). The animal experiments inthis study were approved by the Animal Care and Use Committee of the
NIG.
Sequence analysis

For sequence alignment and homology comparisons, we used the ClustalW
system (http://www.ddbj.nig.ac.jp/search/clustalw-j.html), VISTA program(http://genome.lbl.gov/vista/index.shtml), UCSC genome Browser Home
(http://genome.ucsc.edu/) and Ensemble database (http://www.ensembl.
org/index.html). The draft genome sequences of humans, mice and medaka
fish have been previously described. For homology comparison, we used thefollowing sequences from the UCSC database: mouse Chr5, 28,783,38029,704,930; human Chr7, 155,294,520-156,378,6670; chicken Chr2,
8,031,550-8,431,420; Xenopus Chr28, 4,064,830-4,399,140; medaka
Chr20, 17,738,740-17,852,820. The medaka genome sequence is also
referenced from the NIG DNA sequence center (http://dolphin.lab.nig.
ac.jp/medaka/index.php). The sequence data of the CNCSs MRCS1,
MFCS3, MFCS4, MACS1 and MFCS2 have been submitted to DDBJ under
Accession Numbers AB453051, AB453050, AB258402, AB453049 andAB453052, respectively. MFCS3 and MFCS4 are mouse orthologs of thepreviously described fugu or human sequences,SHH2 (Accession Number
CR847489) and SHH1 (Accession Number CR847488) (Woolfe et al.,2005). The genomic positions of CNCSs in different species, which wereused for the Vista analysis, are listed in Table S1 in the supplementarymaterial.

Development 136 (10)HCl (pH 7.5); 0.1 mM EDTA (pH 8.0)]. DNA (1-4 ng/μl) was purified using
a filter unit and used in injection experiments. Transient transgenic embryos
and stable transgenic mouse lines were generated by pronuclear injection
into fertilized eggs derived from the (C57BL/6⫻DBA/2)F1 or C57BL/6
strain. Transgenic animals were selected using the following primer pairs
for the lacZ gene: F, 5⬘-TCACCCTGCCATAAAGAAACT-3⬘; R, 5⬘CTGTCGTCGTCCCCTCAAACT-3⬘. Whole-mount lacZ staining was
carried out as previously described (Maas and Fallon, 2005). For histologicalanalysis of transgenic embryos, embryos were fixed overnight in 4%
paraformaldehyde, dehydrated in an ethanol series and embedded inparaffin. Sections were cut at 5 μm and counterstained with acidic Fast Red.
ES cell targeting

We used a previously described basic targeting vector to build the MFCS4
targeting construct (Sagai et al., 2005). The long arm (5478 bp) was
amplified from BAC RP23-284A9 DNA with the primer pair 5⬘-ATGGTACCAGGAGATATGCTGCATCCTC-3⬘ and 5⬘-TACTCGAGAGAACTGCGGTTTAACCTGC-3⬘, and the short arm (1824 bp) was amplified
with the primer pair 5⬘-CCGGAATTCGCATTAGAAGCTGGGATGGA-3⬘
and 5⬘-CGCGAATTCGGACCTTACATACGTGAAGC-3⬘. The 999 bp
genomic sequence, including mouse MFCS4, was replaced with the Neo
cassette (see Fig. S1 in the supplementary material). The targeting vector
was electroporated into TT2 ES cells, which originated from a(C57BL/6⫻CBA)F1 mouse (Yagi et al., 1993). ES cells were screened withthe following PCR primer pair: p1, 5⬘-AGTGCTGTCCCAGAGATAAG-3⬘
and p2, 5⬘-CATCGCATTGTCTGAGTAGG-3⬘. Positive clones were
aggregated with eight-cell embryos from (DBA/2⫻C57BL/6)F1 mice andtransplanted into surrogate females. Male chimeras were mated with
C57BL/6 females. Segregation of the targeted allele was determined using
three PCR primers: p2, p3, 5⬘-TCTCAATCTGAACACTGGGC-3⬘, and p4,
5⬘-TCTCAATCTGAACACTGGGC-3⬘. Skeletal analysis of newborn mice
was performed as previously described (Trokovic et al., 2003). For thehistological analysis of the ΔMFCS4/ΔMFCS4 mutants, embryos at E18.5
were preserved in Bouin’s fixative and embedded in paraffin. Serial sectionsof 5 μm were collected and stained with Hematoxylin-Eosin. We referred toan anatomical atlas (Kaufman, 1992) throughout the experiments.
In situ hybridization

For cryosectioning, embryos were fixed in 4% paraformaldehyde andimmersed in 30% sucrose/phosphate-buffered saline overnight, embedded
and frozen in OCT, and sectioned at 15 μm. For paraffin sections, embryos
were fixed with 4% paraformaldehyde and dehydrated in a methanol series,
then embedded in paraffin and sectioned at 8 μm. In situ hybridization was
performed using digoxigenin-UTP-labeled riboprobes, as previously
described (Makino et al., 2001). Whole-mount in situ hybridization was
performed as previously described (Wilkinson, 1992).
RT-PCR

Total RNA was extracted from the anterior tongue, epiglottis-arytenoidswelling and lung tissues of C57BL/6 embryos at E13.5. One μg of each
RNA sample was reverse transcribed into cDNA with SuperScript III
Transcriptase (Invitrogen). One μl of the cDNA solution was used for PCR
amplification. Details of primer pairs used can be provided on request.

Mouse genomic DNA fragments, including the CNCSs were amplified fromRP23-284A9 or RP23-428P20 BAC DNA. After sequencing, the amplified
fragments were inserted into the HindIII or SalI site of the hsp68/LacZ
expression vector (Shashikant et al., 1995). Details of the primer pairs used
for amplification of the inserts can be provided on request. To obtain the
MFCS4 fragment lacking a 217 bp ultra-conserved sequence, inverted tailto-tail primer pairs were used to amplify a basic whole vector, except for the217 bp ultra-conserved sequence of MFCS4. The primer pair used was: F,
5⬘-AGATTGGGTTCACTGTGTGC-3⬘; R, 5⬘-CACAAGCCTCTTTAGTCAGG-3⬘. Then, the deleted form of MFCS4 (ΔMFCS4) was subcloned
into the lacZ reporter construct. The XhoI and NotI double-digested
fragments were cut out from an 0.8% low melting agarose gel and digested
with GELase enzyme (Epicentre Technologies, Madison, WI, USA) at 43°C
overnight. After phenol and chloroform extraction, DNA was precipitatedwith ethanol and dissolved in a small volume of injection buffer [5 mM Tris-

RESULTS
Exploration of novel CNCSs 600-900 kb upstreamof the Shh transcriptional start site
We compared the mouse genomic sequence 600-900 kb upstream of
the Shh transcriptional start site with the syntenic regions of various
tetrapod species and teleost fishes, using public genome databases.
Previously, we have identified three CNCSs in this region that areconserved between mammals and teleost fishes and that we called
Mammal Fish Conserved Sequence 1 to 3 (MFCS1 to 3) (Fig. 1)(Sagai et al., 2005). Here, we describe the identification of three
additional CNCSs in this region (Fig. 1A) that are evolutionarily
conserved at different level (Fig. 1B). MFCS4 is conserved between
mammals and teleost fishes, albeit with slightly lower sequence

DEVELOPMENT

Transgenic assay

Clustering long-range Shh enhancers

RESEARCH ARTICLE 1667
Fig. 1. Location of CNCSs in the interval
between the Shh-coding sequence andLmbr1. (A) The genomes of five evolutionarily
distant species: mouse (chr5), human (chr7),chicken (chr2), Xenopus (chr28) and medaka(chr20). The scale is in kb and is expanded to 2.5fold in the chicken and Xenopus genomes, and10-fold in the medaka genome compared withthe mammalian genomes. To compare the relative
positions of the CNCSs, MFCS1 was right-aligned
at the same position for all species. The coloredovals indicate the locations of MRCS1 (blue),
MFCS3 (orange), MFCS4 (red), MACS1 (green),
MFCS2 (purple) and MFCS1 (yellow). Putative
medaka MACS1 is depicted with a white oval
rimmed with green. (B) VISTA plots of the six
CNCSs. Mouse CNCSs are compared with thehomologs of the human, cow, opossum, platypus,
chicken, lizard, Xenopus, fugu and medaka
CNCSs. Constraints used are a 100 bp window
length and 70% conservation level.

Transgenic reporter assay of the CNCSs
To test the function of the newly identified CNCSs, we carried outtransgenic assays by examining β-galactosidase activity in mouseembryos carrying a ~1 kb mouse genomic sequence containing thefive different CNCSs linked to the lacZ reporter gene. We firstexamined reporter gene expression in whole-mount transgenic
embryos at embryonic day (E) 9.0-15.5. As summarized in Table 1,
MFCS3, MFCS4, MRCS1 and MACS1 directed reproducible
tissue-specific reporter gene expression, whereas MFCS2 did notshow tissue-specific expression. MFCS3 drives lacZ expression in
the brain and motor neurons at E11.5 (see Fig. S2 in the

supplementary material), whereas the other three CNCSs (MFCS4,
MRCS1 and MACS1) drive expression in different epithelial
linings.
MRCS1 drives strong lacZ expression in the epithelia of theincisor and molar teeth at E12.5 (Fig. 2A,B) and weak expression in
the lingual papillae (Fig. 2B). At E13.5, lacZ signal appears in thewhisker buds, tooth primordia, the rugae of the hard palate (Fig. 2C).
Expression in the fungiform papilla of the anterior tongue also
becomes more prominent at this stage (Fig. 2D). Expression in these
domains continues at least until the palatal structure is established at
E15.5 (Fig. 2E-G). At E14.5-15.5, the reporter signals were also
detected in the hair and nail buds (Fig. 2E).
In the MFCS4 transgenic mice, very weak signal was detected in
the oral epithelium at E11.0 (data not shown). At E11.5, weak andtransient signal was detected in the dental germ (Fig. 2H). Strong
expression was observed in the pituitary fossa region and in the
tympanic tube and recess (Fig. 2H). In the lower facial area,

Table 1. lacZ reporter expression directed by CNCSs in transgenic
mice
Number of embryos with specificreporter expression/
total number of embryos examined

Motor neuron
Tooth, lingual papilla
Epiglottis, soft palate
Lung, urogenital organ

MFCS2

MFCS3

MFCS4

MRCS1

MACS1

–
–
–
–

10/13
–
–
–

–
–
18/20
–

–
9/10
–
–

–
–
–
7/10

DEVELOPMENT

similarity than MFCS1-3 (Fig. 1A,B), and is an ortholog of ahuman-fugu conserved sequence called SHH1, which has beenreported by another group (Goode et al., 2005; Woolfe et al., 2005).
Mammal Reptile Conserved Sequence 1 (MRCS1) is conserved
among mammals, chicken and lizard, but not in Xenopus and teleostfishes. Mammal Amphibian Conserved Sequence 1 (MACS1) isconserved among mammals, chicken, lizard and Xenopus, and hasvery weak similarity to a short sequence in the medaka genome (Fig.
1B). MACS1 is a partial cDNA fragment previously registered as
AK043126 in the FANTOM data set (Carninci and Hayashizaki,1999) and is located in intron 8 of Rnf32, a gene with unknown
function.
Thus, in total there are six CNCSs presently known to reside in
the 600 to 900 kb region upstream of the Shh transcriptional start site
(Fig. 1). Notably, the order and orientation of these CNCSs relative
to direction of Shh transcription are conserved across evolutionarily
distant species.

1668 RESEARCH ARTICLE

Development 136 (10)Fig. 2. lacZ reporter expression driven by MRCS1 andMFCS4 in transgenic mouse embryos. (A-G) Reporter
signal in MRCS1 transgenic embryos. X-gal staining is
detected in the dental placode of the maxilla (A) andmandible (B) at E12.5. Signal is also detected in thefungiform papillae (B), whisker buds, teeth, palatal rugae
and fungiform papillae (C,D) at E13.5. At E15.5,
expression is detected in the whisker, hair and nail buds(E); teeth, primary palate and palatal rugae (F); molar teeth
and fungiform papillae (G). (H-M) Reporter signal in
MFCS4 transgenic embryos. (H) At E11.5, transient signal
is detected in the dental placode, and intense signal
around the pituitary fossa, the tympanic tube and recess.
Punctate signal is detected on the tongue surface (inset in
H). (I) At E13.5, expression is detected in the soft palataledge and tympanic tube in the skull base. (J) Strong signal
is detected in the epiglottis and arytenoid swelling. (K,L) At
E15.0, expression is observed in the midline of the soft
palate (K), but the intensity of the signal is depressed in
the epiglottis and arytenoid swelling (L). (M) A mid-sagittal
section of an E13.5 embryo shows reporter signal in theepithelia of the tongue, epiglottis, arytenoid swelling anddorsal pharyngeal wall. a, arytenoid swelling; d, dental
placode; e, epiglottis; it, incisor tooth; fp, fungiform
papillae; mt, molar tooth; n, nail bud; pf, pituitary fossa;
pp, primary palate; r, palatal rugae; s, soft palate; t,tongue; tr, trachea; tt, tympanic tube; w, whisker bud.

arytenoid swelling (Fig. 2L). Histological analysis showed that
MFCS4-driven expression is restricted to the epithelial lining (Fig.
2M).
MACS1 drives lacZ reporter expression in the gut endoderm andcaudal region at E9.0 (Fig. 3A). At E9.5, intense signal marks theregion where the future respiratory tube will evaginate from theprimitive gut (Fig. 3B). As shown in the sagittal section, signal was
detected throughout the continuous lining of the gut endoderm at
E9.5 (Fig. 3C). At E10.5, signal was observed in the gut and cloaca(Fig. 3D), as well as the primary lung buds (Fig. 3E). At E10.5,

Fig. 3. MACS1-driven lacZ reporter expression intransgenic embryos. (A) lacZ reporter expression isobserved throughout the entire gut tube at E9.0. (B) The
condensed signal is observed in the tracheal diverticulum at
E9.5. (C) A mid-sagittal section at E9.5 shows that signal isconfined to the epithelial lining of the endoderm. (D) At
E10.5, strong caudal expression disappears and signal isdetected in the gut endoderm and cloaca. (E) Clear signal isdetected in the primary lung buds. (F) A mid-sagittal sectionof an E10.5 embryo shows reporter signal in the epithelia ofthe ventral pharyngeal wall, laryngotracheal tube andcloaca. (G,H) At E11.5, reporter signal is widely observed in
the laryngotracheal tube, lung, gut (G) and genital tubercle(H). (I) A mid-sagittal section of an E12.5 embryo shows
strong signal in the epithelia of the laryngotracheal tube
and lung. c, cloaca; g, gut; gt, genital tubercle; l, lung; lb,lung bud; lt, larybgotracheal tube; s, stomach; td, tracheal
diverticulum.

DEVELOPMENT

punctuate expression was detected on the tongue surface, but not in
the fungiform papillae (inset in Fig. 2H). lacZ expression persists inthese regions until E13.5 (Fig. 2I,J). Notably, signal was detected in
the palatal shelves of the soft palate, but not in the anterior half ofthe palate or in the hard palate (Fig. 2I), where MRCS1 drives
reporter expression (Fig. 2C,F). Strong expression was detected in
the epiglottis and arytenoid swelling at E13.5 (Fig. 2J), which areessential for respiratory and swallowing functions. At E15.0, palatalformation is completed and signal was observed in the midline of
the soft palate (Fig. 2K), but was decreased in the epiglottis andClustering long-range Shh enhancers

RESEARCH ARTICLE 1669

Fig. 4. Shh is expressed in the epithelial linings along the anteroposterior axis. (A-H) Endogenous Shh and Ptch1 expression in sagittal(A,B,E,F) and cross (C,D,G,H) sections of wild-type embryos. At E13.5, Shh expression is widely detected in the epithelia of the teeth, palatal shelf,
lingual papillae, epiglottis, arytenoid swelling, pharyngeal wall, lung, gut and urogenital tract (A,C). Ptch1 expression is mainly detected in themesenchyme adjacent to the Shh-expressing epithelia (B,D). (E-L) Magnified images in the pharyngeal apparatus. Shh expression is detected in theepithelia of the epiglottis, arytenoid swelling and laryngotracheal tube (E,G). Ptch1 expression is mainly observed in the mesenchyme adjacent to
the Shh-expressing epithelia (F,H). (I-L) Reporter expression in MFCS4 and MACS1 transgenic embryos. MFCS4 reporter expression (I,K) partiallyoverlapped with MACS1-driven expression (J,L) in the epithelia of the arytenoid swelling. (M) Reporter expression in the transgenic mice with atandem array of MFCS4 and MACS1. The linked sequence drives the signal in both expression domains by MFCS4 and MACS1. a, arytenoidswelling; b, bladder; e, epiglottis; it, incisor tooth; in, intestine; l, lung; la, laryngeal aditus; lp, lingual papillae; lt, laryngotracheal tube; mt, molar
tooth; n, nasal septum; p, pharynx; ps, palatal shelf; t, tongue; tr, trachea; u, urethra; uc, umbilical cord.

Comparison of lacZ reporter expression withendogenous Shh expression in the epithelial
linings
We observed endogenous expression of Shh and its downstream
gene Ptch1, and compared it with lacZ reporter expression (Fig. 4).
Endogenous Shh expression is observed in the epithelia of the toothprimordia, lingual papillae, palate, epiglottis, arytenoid swelling,lung, gut and urogenital tracts at E12.5-13.5 (Fig. 4A,C,E,G),
whereas Ptch1 endogenous expression is mainly observed in themesenchyme of the tooth primordia, tongue, epiglottis, arytenoidswelling, lung and gut at these stages (Fig. 4B,D,F,H). As describedabove, the reporter signals driven by MRCS1, MFCS4 and MACS1
are confined to the epithelial linings (Fig. 2M; Fig. 3C,F,I), andmirror the endogenous Shh expression domains. Even though the
MFCS4 and MACS1 expression domains partially overlap at theirborders in the arytenoid swelling (Fig. 4I-L), they are distinct, with
MFCS4 driving expression in the epiglottis (Fig. 2M; Fig. 4I) andMACS1 driving expression in the laryngotracheal tube (Fig. 4J,L).
The MFCS4 and MACS1 tandem construct drives reporter

expression throughout the pharyngeal apparatus and the respiratoryand digestive tubes (Fig. 2M; Fig. 3I; Fig. 4M). Thus, these three
CNCSs appear to contain enhancers that together recapitulate theendogenous pattern of Shh expression in the epithelial linings.
Generation of MFCS4 KO mouse mutant
To examine whether the CNCSs act as functional elements in vivo,
we performed gene targeting, focusing on MFCS4. Following thesame strategy used to generate MFCS1 knockout mice (Sagai et al.,2005), we replaced a ~1 kb genomic region, including MFCS4 witha neo cassette. We successfully generated two lines of germline
chimeras, then, heterozygotes (ΔMFCS4/+) were intercrossed togenerate homozygous mutants (ΔMFCS4/ΔMFCS4) (Table 2).
ΔMFCS4/ΔMFCS4 mice were present at E18.5 and early postnatal
stages, but all of them died within a few days of birth (Table 2).
Although ΔMFCS4/+ mice are viable and grossly indistinguishable
from wild-type mice, many ΔMFCS4/ΔMFCS4 neonates have
Table 2. Viability of MFCS4 KO mouse mutants
Number of mice with MFCS4 genotype
Age

E18.5
P0
P1
P2
1-2 months

+/+

+/–

–/–

22
6 (1)*
15
4
37

40
10
28
10
71

11
8 (4)*
12 (10)*5 (5)*
0*In parentheses is the number of mice that died on the day.

DEVELOPMENT

intense signal was apparent in the epithelia of the laryngo-tracheal
tube and cloaca. At E11.5, strong expression was observed in thelaryngotracheal tube, lung, gut (Fig. 3G) and genital tubercle (Fig.
3H). A mid-sagittal section confirmed the intense signal in theepithelia of the laryngotracheal tube, lung, digestive tube andurogenital tract (Fig. 3I; data not shown) at E12.5. Thus, MACS1
strictly specifies the expression in the epithelia of the respiratory anddigestive tubes, which are derived from the primitive gut endoderm.

1670 RESEARCH ARTICLE

Development 136 (10)bloated bellies, which might be caused by air accumulation in thestomach and bowels (Fig. 5A), and their stomachs are always devoidof milk (Fig. 5B). The tongue, soft palate, epiglottis and arytenoid
comprise the pharyngeal apparatus, which is essential for respirationand swallowing (Fig. 5C,D,F,G). In the ΔMFCS4/ΔMFCS4 mice,truncation of the soft palate (Fig. 5E), loss or reduced size of theepiglottis and hypotrophy of the arytenoid (Fig. 5H,I), and tongue
deformation (Fig. 5H) are observed. Moreover, deletion of MFCS4
appears to result in minor morphological abnormalities of thebasisphenoid and basioccipital bones, as is visible from a ventral
view of the skull (Fig. 5J,K). The cartilage between these bones,
which is probably the pituitary fossa region that closes after
formation of the pituitary, has a hole (marked with the broken whiteline in Fig. 5K). The ΔMFCS4/ΔMFCS4 mice display defects in thehyoid bone and thyroid cartilage (Fig. 5L,M). Although there isvariability in the severity of the defects, the major phenotype wascompletely penetrant, with abnormalities observed in the
aforementioned structures in all the ΔMFCS4/ΔMFCS4 mice
examined (n=17).
Histological analysis of the sagittal sections showed that the
ΔMFCS4/ΔMFCS4 embryos have deformed posterior tongues,
hypoplasia of the hyoid cartilage, loss of the epiglottis, hypoplasiaof the arytenoid and shortening of the soft palate (Fig. 5O), which
eventually resulted in atypical junctions between the nasopharynx
and oropharynx. The posterior tongue edge, including hyoid bone
and thyroid cartilage was deformed (Fig. 5M,O), and the base of

DEVELOPMENT

Fig. 5. Phenotypes of ΔMFCS4/ΔMFCS4 mice. (A) Many ΔMFCS4/ΔMFCS4 mice had a dilated belly (asterisk). (B) A ΔMFCS4/ΔMFCS4 neonate
whose stomach is not filled with milk (asterisk) (B). (C-M) Phenotypes of embryos at E18.5. (C) Wild-type oro- and naso-pharyngeal junction, upper
right. The epiglottis and arytenoid are clearly recognizable. (D,E) Lower aspect of the hard and soft palates, and the naso-pharyngeal opening. In
wild-type mice, the hard palate (hp, double arrow) and soft palate (sp, double arrow) are well formed (D). In ΔMFCS4/ΔMFCS4 mice, the palates are
fused at the midline. The hard palate is formed normally, but the soft palate terminates prematurely. As a consequence, the naso-pharyngeal
opening in ΔMFCS4/ΔMFCS4 mice is misaligned to the anterior side (E). (F-I) Views of the dorsal aspect of the tongue and the oro-pharyngeal
opening. (G,I) Magnified images of the oro-pharyngeal opening. In ΔMFCS4/ΔMFCS4 mice, the tongue is misshapen (H) compared with wild-type(F), the epiglottis and arytenoid are hypoplastic (I) compared with wild-type (G). (J,K) Ventral view of the skull. Mutant animals have minor
morphological anomalies in the basisphenoid and basioccipital bones, and have a hole in the spheno-occipital synchondrosis just inferior to thepituitary (K). (L,M) Dorsal views of the laryngeal cartilages. In mutant animals, the hyoid bone is malformed and the thyroid cartilage appeared to behypoplastic (M). (N,O) Mid-sagittal sections of the oral and pharyngeal structures of heterozygous and homozygous mutant animals at E18.5. In
ΔMFCS4/+ mice, the posterior edge of the soft palate extends alongside the posterior pharyngeal wall and is situated close to the epiglottis. The
epiglottis and arytenoid are well formed (N). By contrast, in ΔMFCS4/ΔMFCS4 mice, the soft palate terminates prematurely, and the posterior
tongue, epiglottis and arytenoid are severely deformed. The cross-sectional diameter of the body of the hyoid bone is decreased (thinner), and thebone and cartilage beneath the pituitary gland show morphological abnormalities (O). Red outline indicates the affected area in the
ΔMFCS4/ΔMFCS4 mutant. a, arytenoid; bo, basioccipital bone; bs, basisphenoid; cc, cricoid cartilage; e, epiglottis; es, esophagus; hb, hyoid bone;
hp, hard palate; it, incisor tooth; m, mandible; p, palate; pg, pituitary gland; sp, soft palate; t, tongue; tc, thyroid cartilage; tr, tracheae.

Clustering long-range Shh enhancers

RESEARCH ARTICLE 1671

Fig. 6. MFCS4 is a cis-acting regulator of Shh. (A-D) Shh expression in wild-type and ΔMFCS4/ΔMFCS4 embryos. (A) In wild-type embryos, Shhexpression is detected in the epiglottis and arytenoid swelling at E13.5. (B) In ΔMFCS4/ΔMFCS4 embryos, Shh expression is lost in both structures.
(C,D) Section in situ hybridization for Shh in wild-type and ΔMFCS4/ΔMFCS4 embryos at E13.0. (C) In wild-type embryos, Shh signal is broadly
detected in the pharyngeal epithelium. (D) In ΔMFCS4/ΔMFCS4 embryos, Shh expression is downregulated in the epithelium of the pharyngeal wall,
which is the future oro-pharynx area, and expression almost completely disappears in the epithelium of the epiglottis, the thyroid duct and thearytenoid swelling. Shh signal is detected at wild-type levels in the incisor tooth primordium of ΔMFCS4/ΔMFCS4 embryos (D). Boxed areas indicate
Shh expression in the pharyngeal region of the wild type and the ΔMFCS4/ΔMFCS4 mutant. (E-H) Genetic test for cis-activity of MFCS4. (E) Diagramshows how cis or trans regulation of Shh expression by MFCS4 would function in the pharyngeal epithelium. If MFCS4 were a trans-acting
regulator of Shh expression, an intact MFCS4 sequence located in a cis position relative to the Shh KO allele should be able to activate transcriptionof the intact Shh gene on the trans allele. Therefore, the ΔMFCS4/ΔMFCS4 phenotype should be rescued in Shh KO: MFCS4 KO compound
heterozygotes. Conversely, if MFCS4 were a cis-acting regulator, the intact MFCS4 sequence would not be able to activate transcription of the Shhgene on the trans allele, and the compound heterozygotes would have the same phenotype as individuals with the single MFCS4 mutation.
(F-H) Phenotypes of E18.5 embryos from the cross mating of Shh+/– mice to ΔMFCS4/+ mice. The four expected genotypes of the progeny
segregated in a Mendelian fashion. Among them, the wild-type and the Shh+/– or ΔMFCS4/+ mice had normal phenotypes with respect to theoro-pharyngeal openings (G). However, all compound heterozygotes had abnormal openings, as did single ΔMFCS4/ΔMFCS4 mutants (H).
a, arytenoid swelling; e, epiglottis; it, incisor tooth; m, mandible; t, tongue.

expression in the epithelia of the epiglottis and arytenoid swelling(Fig. 6B,D). However, Shh expression in the tooth primordia was
not altered in the mutant embryos (Fig. 6D), in accordance with thenormal tooth development in the ΔMFCS4/ΔMFCS4 embryos (Fig.
5O).
Finally, we carried out a cis-trans test by mating animals
heterozygous for the Shh-coding sequence KO allele (Shh+/–) to the
ΔMFCS4/+ mouse. All compound heterozygotes exhibited
hypoplasia of the epiglottis and arytenoids, indicating that MFCS4
is cis-acting to the Shh-coding sequence (Fig. 6E,F). Together, ourresults indicate that MFCS4 is a pharyngeal epithelium-specificenhancer of Shh, and is indispensable for morphogenesis of thepharyngeal structures in the mouse.
Evolutionary rigidity assay (ERA) of MFCS4
Alignment of the MFCS4 nucleotide sequences across distantspecies revealed a 217 bp ultra-conserved sequence, which isconserved from mammals to teleost fish medaka (Fig. 1A; see Fig.
S3 in the supplementary material). To assess importance of thesequence for the MFCS4 enhancer activity, we carried out atransgenic assay for the lacZ reporter construct, in which the MFCS4
fragment lacks the 217 bp sequence (see Fig. S3 in the
supplementary material). The result showed that this deletion formof MFCS4 drives no reporter expression in the relevant tissues of thetransgenic embryos (0/5). It supports the fact that the 217 bp ultra-

DEVELOPMENT

tongue seemed to be affected as well, which eventually result in theatypical tongue (Fig. 5H). Though the transgenic MFCS4 reporter
signal was detected in the tympanic tube and recess, visible defects
were not obvious in the auditory organs of the ΔMFCS4/ΔMFCS4
neonates. Most of the ΔMFCS4/ΔMFCS4 had normal hard palate,
and a few neonates (5/37) exhibited cleft palate (data not shown).
Notably, in the ΔMFCS4/ΔMFCS4 neonates, the tooth and whisker,
in which the MFCS4-mediated lacZ expression was not observed,
but the MRCS1-mediated lacZ expression was observed, were notaffected (inset in Fig. 5O; data not shown). As described before, thereporter expression domains driven by MFCS4 and MACS1 arepartially overlapped in the arytenoid swelling. It is consistent withthe fact that the posterior border of the defects observed in the
ΔMFCS4/ΔMFCS4 neonates corresponds to the posterior end of thepharynx. The ΔMFCS4/ΔMFCS4 neonates showed no visible
defects in the cricoid cartilage, trachea, lung, esophagus, digestive
tube and urogenital organs (see Table S2 in the supplementarymaterial). Thus, MFCS4 is crucial for development specifically of
the pharyngeal apparatus.
We next examined whether the Shh pathway is altered in the
ΔMFCS4/ΔMFCS4 embryos by whole-mount and section in situ
hybridization using a Shh riboprobe. Strong Shh expression was
detected in the epithelium of the epiglottis and arytenoid swelling of
wild-type embryos at E13.0 and E13.5 (Fig. 4A,C; Fig. 6A,C),
whereas the ΔMFCS4/ΔMFCS4 embryos lost almost all Shh1672 RESEARCH ARTICLE

Development 136 (10)conserved sequence is indispensable for the MFCS4 enhancer
activity. To explore transcription factors that bind to this sequence,
we carried out the evolutionary rigidity assay (ERA) for the 217 bp
sequence. We identified four nearly perfect match motifs (NPMMs)(see Fig. S3 in the supplementary material), which are potentiallybinding sites for Pbx1 (MFCS4-MF-A), Sox5 (MFCS4-MF-B),
TCFs (MFCS4-MF-C), inner-cell mediators of Wnt signaling and
Hes1 (MFCS4-MF-D), a well known target of Notch signaling.
A number of reports suggest that Pbx, Sox, Wnt and Notch
signaling pathways are indispensable for normal development ofepithelial tissues such as tooth, hair follicle, taste papillae, lung andgastrointestinal tract (Ito et al., 2000; Iwatsuki et al., 2007; Li et al.,2005; Okubo et al., 2006; Schnabel et al., 2001). We examined
expression of many genes involving these signaling pathways inthe tongue, epiglottis and lung. As shown in Fig. S4 in the
supplementary material, most of the genes are activated in theepiglottis and arytenoid swelling at E13.5. The results suggested thatthey are good candidates for the MFCS4 activity.

Fig. 7. Genomic map of Shh enhancers. (A) The 840 kb genome
region upstream of the Shh transcriptional start site contains numerous
regulatory elements. Previously described enhancers directing Shh
expression in the CNS are also shown (Jeong et al., 2006).
(B) Schematic diagram showing the expression domains of Shh inepithelial lining regulated by MRCS1 (blue), MFCS4 (red) and MACS1(green) along the anteroposterior axis.

is necessary for the formation of the lung, gut and urogenital organs(Haraguchi et al., 2007; Litingtung et al., 1998). Our transgenic
experiments showed that timing of reporter expression onset driven
by MRCS1, MFCS4 and MACS1 in each lining is consistent withthe endogenous temporal expression pattern of Shh, suggesting thatthe three enhancers identified in this study regulate endogenous Shh
expression. However, it is notable that the co-linearity in thetemporal expression patterns driven by the three enhancers is not as
obvious as that observed for the regional expression patterns.
At present we have no compelling interpretation for the biologicalimplication of the long-range enhancers that regulate the Shh
expression. Our very recent study (Amano et al., 2009)
demonstrated that chromosome dynamics is involved in Shh
expression in the mouse developing limb bud. A long-range limb
bud-specific enhancer, MFCS1, specifically interacts with the Shhpromoter via change of the chromosome conformation. The Shh
expression regulated by the epithelial lining-specific enhancers
identified in this study is probably exerted by the similar mechanism.
Therefore, it is of interest to investigate how the co-linear Shh
expression in the epithelial linings along the anteroposteior body
axis is regulated by orchestrated chromosome dynamics.
Indispensable role of MFCS4 in morphogenesis ofpharyngeal apparatus
The anterior endoderm-derived pharynx is the region of thedigestive tube anterior to the point where the respiratory tube
branches off. Loss-of-function and misexpression experiments have

DEVELOPMENTDISCUSSION
Three clustered enhancers direct co-linear Shh
expression along the anteroposterior axis in theepithelial linings
In this study, we have identified three novel CNCSs in the region
620 to 740 kb upstream of the Shh transcriptional start site. Theirsequences, order and orientation are highly conserved between
evolutionarily distant species (Fig. 1A; Fig. 7A). These CNCSs
direct regional expression of Shh in the epithelial linings from theoral cavity to the hindgut along the anteroposterior body axis (Fig.
7B). At the most anterior extent, MRCS1 directs Shh expression in
the epithelia of the hair and whisker buds, dental placode, rugae ofthe hard palate and fungiform papillae of the anterior tongue, all ofwhich are presumptive derivatives of the oral ectoderm. MFCS4
drives expression in the soft palate, epiglottis, arytenoid swellingand other pharyngeal tissues, forming a border with the moreanterior MRCS1-directed expression domain. Finally, MACS1
drives expression in the respiratory and digestive tubes, with theanterior border of expression partially overlapping the posteriorborder of MFCS4-driven expression in the pharyngeal structures.
Thus, this study clearly shows that three distinct enhancers regulate
Shh expression along the long continuous epithelial linings from theoral cavity to the hindgut. Interestingly, the genomic order of thesethree enhancers is co-linear with the regional control of Shh
expression along the anteroposterior body axis (Fig. 7). Thissuggests that, at least with regard to the Shh pathway, morphogenesisof the epithelial linings is roughly partitioned into three components:
the oral ectoderm, pharyngeal endoderm and gut endoderm. At
present, the biological implications of the co-linearity of theseenhancers are uncertain, but it is possible that clustering of the threeenhancers is required for the proper spatiotemporal regulation of Shh
expression.
Endogenous Shh expression is initiated progressively along thecontinuous epithelial linings spanning from the oral cavity to thehindgut (Bitgood and McMahon, 1995; Iseki et al., 1996; Varjosalo
and Taipale, 2008). In the anterior ectoderm-derived epithelial
lining, Shh expression starts around E11.5-12.5 and is implicated indevelopment of the oral cavity, the tooth and the tongue (Cobourne
et al., 2004; Hall et al., 1999). At E11.5-12.5, the pharyngeal arches
become broadened and flattened externally, and form the neck of theembryo. Around this stage, Shh expression is observed in thepharyngeal epithelial lining (Fig. 4E,G) (Rice et al., 2006). In the
primary gut, the earliest Shh expression is detected around E8.0 andshown that the Shh pathway functions in the pharyngeal endoderm
to generate multiple organs, including the pancreas, pituitary gland,parathyroid gland and jaw (Brito et al., 2006; Hebrok, 2003;
Litingtung et al., 1998; Sbrogna et al., 2003; Treier et al., 2001).
However, owing to severe abnormalities in the Shh KO mutant
mouse, the function of Shh at later stages of the pharyngeal
morphogenesis has been poorly understood. In this study, we
examined a functional requirement for the pharyngeal epitheliumspecific Shh enhancer, MFCS4, by targeted deletion of MFCS4 inmice. The result clearly showed that MFCS4 is essential formorphogenesis of the pharyngeal structures necessary for respirationand swallowing, including the soft palate, epiglottis and arytenoid.
As shown in Table S2 in the supplementary material, relevance
between domains of the reporter expression driven by the threeepithelial Shh enhancers and the phenotype in the
ΔMFCS4/ΔMFCS4 neonates shows a crucial role of the Shhpathway in the morphogenesis of the pharyngeal epithelium.
Moreover, detailed characterization of the phenotype of the
ΔMFCS4/ΔMFCS4 embryos suggested that the Shh pathway is
involved in regional segmentation of the epithelial linings. This will
be confirmed by future studies with knockout mutants of MRCS1
and MACS1.
We found that the 217 bp ultra-conserved sequence of MFCS4
has the four motifs for the transcription factors involved in the Pbx,
Sox, Wnt and Notch signaling pathways. As we revealed that many
genes encoding these transcription factors are expressed in theepiglottis and arytenoid swelling of the E13.5 embryos, in which weobserved the highest level of the MFCS4-mediated lacZ reporter
expression, these transcription factors most probably act as direct
upstream regulators of the Shh expression in the pharyngeal
epithelium.
Here, we need to pay attention to influence of the adjacent genes
Lmbr1 and Rnf32 on the phenotype of the ΔMFCS4/ΔMFCS4
embryos, because the deletion of MFCS4 may disrupt the functionsof these genes. The Lmbr1 null mutation has been reported
previously (Clark et al., 2000). The Lmbr1 KO homozygotes show
limb defects, but they are viable and fertile. It suggests that
disruption of the Lmbr1 gene cannot cause crucial pharyngeal
defects, as shown in the ΔMFCS4/ΔMFCS4 embryos. The Rnf32
mutant mouse has not been reported thus far, and the function of thisgene remains unclear. Thus, influence of the MFCS4 disruption on
the pharyngeal phenotype is currently undeniable.
Evolutionary diversification of the enhancers
Morphological variation in the epithelial architecture is wellexemplified in the evolutionary distant species (Botella et al., 2007;
Brainerd and Owerkowicz, 2006; Delgado et al., 2005; Ichim et al.,2007; Iwasaki, 2002; Mitsiadis et al., 2003). We showed that the oral
epithelium-specific enhancer MRCS1 is conserved in birds andreptiles at the almost same level as in mammals, whereas its
homolog has not been identified in amphibians and teleost fishes.
The lung-gut epithelium-specific enhancer MACS1 is conserved inamphibians, but not in teleost fishes. It is likely that the various
epithelial derivatives evolved from a flat epithelial structure(Chuong and Edelman, 1985; Chuong et al., 2000), and that theirshapes and sizes can be altered, depending on the timing andlocation of Shh signaling. Thus, regulation of Shh expression
probably influences the architecture of the epithelial derivatives. At
present, it is not clear how the oral and lung-gut epithelial
architecture develop in the species without MRCS1 and MACS1.
However, one possible explanation is that, in the past, MFCS4
specified Shh expression in both the ectoderm-derived oral cavity

RESEARCH ARTICLE 1673

and the anterior endoderm-derived pharynx. Indeed, we found thatmouse MFCS4 drives transient reporter expression in the dental
placode (Fig. 2H). Alternatively, there may be unidentified, speciesspecific enhancers that regulate Shh expression in a tissue-specificmanner in amphibians and teleost fishes. It would be of interest to
explore whether such CNCSs exist within different teleost fishes or
between amphibian and teleost fishes.
We are grateful to Drs Y. Katori, T. Kobayashi, S. Iseki and N. Wada for helpfulcomments on anatomy, to Drs T. Takada, S. Tanaka, N. Sakai, M. Shinya, H.
Kokubo and M. Okabe for useful discussion throughout this study, and to Dr S.
Kitajima for technical advice on ES cell manipulation. We thank Ms N.
Yamatani, H. Nakazwa, A. Okagaki and Y. Kato for their kind technicalsupport. We are also grateful to Dr P. Beachy for providing us with the Shhknockout mice, to Dr A. McMahon for providing the Shh probe and to Dr M. P.
Scott for the Ptch1 probe. This study was supported in part by grants-in-aid
from the Ministry of Education, Culture, Sports, Science and Technology ofJapan. This paper is contribution number 2513 from the National Institute of
Genetics, Japan.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/cgi/content/full/136/10/1665/DC1
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