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First published online 15 April 2009
doi: 10.1242/dev.032714
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1 Mammalian Genetics Laboratory, National Institute of Genetics, Yata-1111
Mishima Shizuoka-ken 411-8540, Japan.
2 Population Genetics Laboratory, National Institute of Genetics, Yata-1111
Mishima Shizuoka-ken 411-8540, Japan.
* Author for correspondence (e-mail: tshirois{at}lab.nig.ac.jp)
Accepted 10 March 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Sonic hedgehog, Conserved non-coding sequence, Long-range enhancer, Epithelial lining, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
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) and
anterorposterior 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 of mature morphologies are
exhibited (Chuong et al.,
2000). Targeted deletion of Shh has shown a role for
Shh signaling in the development 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 in later 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;
Goode et al., 2005;
Santagati et al., 2003;
Woolfe and Elgar, 2007;
Woolfe et al., 2007;
Woolfe et al., 2005).
Functional analysis of these elements has been carried out using BAC reporter
transgenesis in mice (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 observed in the KO mutant of the Shh-coding sequence
(Chiang et al., 1996).
Subsequent cis-trans tests verified that the CNCS contains a limb-specific
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 gene density 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 and transgenic 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 of
holoprosencephaly (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 the transcriptional 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 not been reported.
Here, we explore new CNCSs in the mouse 300 kb genomic region 600 to 900 kb upstream of the Shh-coding sequence by comparing the mouse genome with the genomes of other mammalian species and teleost fishes. We identify a cluster of three CNCSs that drive lacZ reporter expression in the epithelia of the oral apparatus, the pharyngeal apparatus, and the lung and gut. Interestingly, the co-linear genomic arrangement of the three CNCSs mirrors the anteroposterior order of their expression domains, partitioning the continuous epithelial lining into three Shh expression domains: the ectoderm-derived oral cavity, the anterior endoderm-derived pharynx, and posterior 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.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 the following sequences
from the UCSC database: mouse Chr5, 28,783,380-29,704,930; 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 and AB453052, respectively. MFCS3 and MFCS4 are mouse orthologs of
the previously 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 were used for the Vista
analysis, are listed in Table S1 in the supplementary material.
Transgenic assay
Mouse genomic DNA fragments, including the CNCSs were amplified from
RP23-284A9 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 tail-to-tail primer pairs were used to
amplify a basic whole vector, except for the 217 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
(
MFCS4) 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 precipitated with ethanol and dissolved in a
small volume of injection buffer [5 mM Tris-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/6xDBA/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
histological analysis of transgenic embryos, embryos were fixed overnight in
4% paraformaldehyde, dehydrated in an ethanol series and embedded in paraffin.
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/6xCBA)F1 mouse
(Yagi et al., 1993). ES cells
were screened with the following PCR primer pair: p1,
5'-AGTGCTGTCCCAGAGATAAG-3' and p2,
5'-CATCGCATTGTCTGAGTAGG-3'. Positive clones were aggregated with
eight-cell embryos from (DBA/2xC57BL/6)F1 mice and
transplanted 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
the histological analysis of the MFCS4/MFCS4 mutants, embryos at
E18.5 were preserved in Bouin's fixative and embedded in paraffin. Serial
sections of 5 µm were collected and stained with Hematoxylin-Eosin. We
referred to an anatomical atlas (Kaufman,
1992) throughout the experiments.
In situ hybridization
For cryosectioning, embryos were fixed in 4% paraformaldehyde and immersed
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-arytenoid
swelling 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.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). 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
similarity than MFCS1-3 (Fig.
1A,B), and is an ortholog of a human-fugu conserved sequence
called SHH1, which has been reported 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 teleost fishes. Mammal Amphibian
Conserved Sequence 1 (MACS1) is conserved among mammals, chicken, lizard and
Xenopus, and has very 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.
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Transgenic reporter assay of the CNCSs
To test the function of the newly identified CNCSs, we carried out
transgenic assays by examining β-galactosidase activity in mouse embryos
carrying a 
1 kb mouse genomic sequence containing the five different
CNCSs linked to the lacZ reporter gene. We first examined 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 not show
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.
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In the MFCS4 transgenic mice, very weak signal was detected in the oral epithelium at E11.0 (data not shown). At E11.5, weak and transient 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, punctuate expression was detected on the tongue surface, but not in the fungiform papillae (inset in Fig. 2H). lacZ expression persists in these 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 of the 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 are essential for respiratory and swallowing functions. At E15.0, palatal formation is completed and signal was observed in the midline of the soft palate (Fig. 2K), but was decreased in the epiglottis and arytenoid swelling (Fig. 2L). Histological analysis showed that MFCS4-driven expression is restricted to the epithelial lining (Fig. 2M).
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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 the same strategy used
to generate MFCS1 knockout mice (Sagai et
al., 2005), we replaced a 1 kb genomic region, including
MFCS4 with a neo cassette. We successfully generated two lines of
germline chimeras, then, heterozygotes (MFCS4/+) were intercrossed to
generate homozygous mutants (MFCS4/MFCS4)
(Table 2).
MFCS4/MFCS4 mice were present at E18.5 and early postnatal
stages, but all of them died within a few days of birth
(Table 2). Although
MFCS4/+ mice are viable and grossly indistinguishable from wild-type
mice, many MFCS4/MFCS4 neonates have bloated bellies, which
might be caused by air accumulation in the stomach and bowels
(Fig. 5A), and their stomachs
are always devoid of milk (Fig.
5B). The tongue, soft palate, epiglottis and arytenoid comprise
the pharyngeal apparatus, which is essential for respiration and swallowing
(Fig. 5C,D,F,G). In the
MFCS4/MFCS4 mice, truncation of the soft palate
(Fig. 5E), loss or reduced size
of the epiglottis 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 the basisphenoid 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 white line in
Fig. 5K). The
MFCS4/MFCS4 mice display defects in the hyoid bone and thyroid
cartilage (Fig. 5L,M). Although
there is variability in the severity of the defects, the major phenotype was
completely penetrant, with abnormalities observed in the aforementioned
structures in all the MFCS4/MFCS4 mice examined
(n=17).
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MFCS4/MFCS4 embryos have deformed posterior tongues, hypoplasia
of the hyoid cartilage, loss of the epiglottis, hypoplasia of 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 tongue seemed to be affected as well, which
eventually result in the atypical 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
MFCS4/MFCS4 neonates. Most of the MFCS4/MFCS4 had
normal hard palate, and a few neonates (5/37) exhibited cleft palate (data not
shown). Notably, in the MFCS4/MFCS4 neonates, the tooth and
whisker, in which the MFCS4-mediated lacZ expression was not
observed, but the MRCS1-mediated lacZ expression was observed, were
not affected (inset in Fig. 5O;
data not shown). As described before, the reporter expression domains driven
by MFCS4 and MACS1 are partially overlapped in the arytenoid swelling. It is
consistent with the fact that the posterior border of the defects observed in
the MFCS4/MFCS4 neonates corresponds to the posterior end of the
pharynx. The MFCS4/MFCS4 neonates showed no visible defects in
the cricoid cartilage, trachea, lung, esophagus, digestive tube and urogenital
organs (see Table S2 in the supplementary material). Thus, MFCS4 is crucial
for development specifically of the pharyngeal apparatus.
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MFCS4/MFCS4 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 MFCS4/MFCS4 embryos lost almost all Shh
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 the normal tooth development in the MFCS4/MFCS4
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 MFCS4/+ 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, our results indicate that MFCS4 is a pharyngeal
epithelium-specific enhancer of Shh, and is indispensable for
morphogenesis of the pharyngeal structures in the mouse.
Evolutionary rigidity assay (ERA) of MFCS4
Alignment of the MFCS4 nucleotide sequences across distant species revealed
a 217 bp ultra-conserved sequence, which is conserved from mammals to teleost
fish medaka (Fig. 1A; see Fig.
S3 in the supplementary material). To assess importance of the sequence for
the MFCS4 enhancer activity, we carried out a transgenic 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 form of MFCS4 drives no reporter expression in the relevant
tissues of the transgenic embryos (0/5). It supports the fact that the 217 bp
ultra-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 potentially binding 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 of epithelial tissues such as tooth,
hair follicle, taste papillae, lung and gastrointestinal 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 in the
tongue, epiglottis and lung. As shown in Fig. S4 in the supplementary
material, most of the genes are activated in the epiglottis and arytenoid
swelling at E13.5. The results suggested that they are good candidates for the
MFCS4 activity.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Endogenous Shh expression is initiated progressively along the
continuous epithelial linings spanning from the oral cavity to the hindgut
(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 in development 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 the embryo. Around this stage,
Shh expression is observed in the pharyngeal epithelial lining
(Fig. 4E,G)
(Rice et al., 2006). In the
primary gut, the earliest Shh expression is detected around E8.0 and
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 with the endogenous
temporal expression pattern of Shh, suggesting that the three
enhancers identified in this study regulate endogenous Shh
expression. However, it is notable that the co-linearity in the temporal
expression patterns driven by the three enhancers is not as obvious as that
observed for the regional expression patterns.
|
) 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 Shh
promoter 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 of pharyngeal apparatus
The anterior endoderm-derived pharynx is the region of the digestive tube
anterior to the point where the respiratory tube branches off.
Loss-of-function and misexpression experiments have 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 epithelium-specific Shh enhancer, MFCS4, by
targeted deletion of MFCS4 in mice. The result clearly showed that MFCS4 is
essential for morphogenesis of the pharyngeal structures necessary for
respiration and 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 three epithelial
Shh enhancers and the phenotype in the MFCS4/MFCS4
neonates shows a crucial role of the Shh pathway in the morphogenesis
of the pharyngeal epithelium. Moreover, detailed characterization of the
phenotype of the MFCS4/MFCS4 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 the epiglottis and arytenoid swelling of the E13.5 embryos, in which we observed 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
MFCS4/MFCS4 embryos, because the deletion of MFCS4 may disrupt
the functions of 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
MFCS4/MFCS4 embryos. The Rnf32 mutant mouse has not
been reported thus far, and the function of this gene 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 well exemplified
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
and reptiles 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 in amphibians, 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 their shapes and sizes can be altered,
depending on the timing and location 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 and the anterior
endoderm-derived pharynx. Indeed, we found that mouse MFCS4 drives transient
reporter expression in the dental placode
(Fig. 2H). Alternatively, there
may be unidentified, species-specific enhancers that regulate Shh
expression in a tissue-specific manner 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.
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Footnotes |
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Supplementary material available online at http://dev.biologists.org/cgi/content/full/136/10/1665/DC1
We are grateful to Drs Y. Katori, T. Kobayashi, S. Iseki and N. Wada for helpful comments 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 technical support. We are also grateful to Dr P. Beachy for providing us with the Shh knockout 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 of Japan. This paper is contribution number 2513 from the National Institute of Genetics, Japan.
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Abbasi, A. A., Paparidis, Z., Malik, S., Goode, D. K., Callaway,
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