Mammalian 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 30 November 2004
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SUMMARY |
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Key words: Limb development, Shh, cis-acting regulator, Medaka, Mouse
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Introduction |
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Use of a comparative genomic approach for identifying functional units
within genomic sequences has increasingly gained support as a result of
several recent reports (Boffelli et al.,
2004). The paired fins of teleost fishes and tetrapod limbs have
evolved from common ancestral appendages
(Grandel and Schulte-Merker,
1998
; Sordino et al.,
1995
; Ruvinsky and
Gibson-Brown, 2000
). Previous phylogenetic studies have revealed
that the intronic sequence of Lmbr1 is highly conserved among
tetrapods and teleost fishes (Sagai et
al., 2004
). In this study, we first examined the physical linkage
of Shh and the conserved intronic sequence in a teleost fish, medaka,
because the genome sequence of the medaka fish is now available. The conserved
sequence in the medaka fish is located in the same scaffold as the medaka
Shh gene, and is placed in intron 5 of the medaka Lmbr1
homologue. This indicates that the physical linkage of the Shh coding
sequence and the conserved intronic sequence evolved prior to the divergence
of teleost fishes and tetrapods.
Based on these data, we wished to examine directly the role of the
conserved sequence in mouse limb development by gene targeting. The knockout
mouse showed a complete loss of Shh expression in the limb buds and
severe amputation of distal elements of the limbs, a phenotype similar to the
Shh KO mouse (Chiang et al.,
1996; Chiang et al.,
2001
) and the human congenital deformity acheiropodia
(Ianakiev et al., 2001
). All
results provided unequivocal evidence that the intronic sequence contains a
major enhancer for limb-specific expression of Shh and is essential
for distal limb development.
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Materials and methods |
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Animals
Two preaxial polydactylous mutants, Hx, M100081, were described
previously (Sagai et al.,
2004). The Shh knockout mouse was kindly provided by Dr
P. Beachy (Chiang et al.,
1996
). The animal experiments in this study were approved by the
Animal Care and Use Committee of National Institute of Genetics.
ES targeting
The basic targeting vector was constructed by inserting pKO Neo and pKO DT
cassettes into the pKO Scrambler V901 vector (Lexicon Genetics Incorporated).
The long arm (5652 bp) and the short arm (2357 bp) fragments derived from BAC
clone BAC311J12 (129/Sv origin) were ligated into the basic vector to replace
the conserved sequence (1167 bp) with the Neo cassette. The targeting vector
was electroporated into R1 ES cells, which originated from the 129/Sv strain.
Recombined cells were screened with the following PCR primer pairs: p1;
5'-TTAAGCATGCTTGTCTCTCG-3' and p2;
5'-CATCGCATTGTCTGAGTAGG-3'. Positive clones were aggregated with
8-cell embryos from (DBA/2x C57BL/6J) F1 mice and transplanted into surrogate
females. Male chimeras were mated with C57BL/6J females, and germline
transmission of the knockout allele was confirmed by presence of the Agouti
coat color. Genotyping of mice was carried out using the following PCR primer
pairs: p3; 5'-GACCAATTATCCAAACCATC-3' and p4;
5'-TAACACTAAGCAGCACTTCC-3', p5;
5'-GGCTATTCGGCTATGACTGG-3' and p6;
5'-GAGATGACAGGAGATCCTGC-3'.
Skeletal preparation
Mouse skeletons were stained by alizarin red and alcian blue as described
previously (Wallin et al.,
1994). For E14 embryos, cartilage was stained as described
previously (Jegalian and De Robertis,
1992
).
Whole-mount in situ hybridization
Whole-mount in situ hybridization of embryos was performed according to the
method described by Wilkinson (Wilkinson,
1992). Briefly, digoxygenin-labeled riboprobes were transcribed in
vitro according to the manufacturer's protocol (Roche). The following probes
were used: Shh (A. McMahon), Gli3 (C. C. Hui), Fgf4
(G. Martin) and dHand (Hand2 Mouse Genome
Informatics) [generated from the entire coding region of murine dHand
(Srivastava et al.,
1995
)].
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Results |
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Disruption of MFCS1 causes severe defects in limb development
We carried out gene targeting experiments to examine directly the role of
MFCS1 in limb development. To generate a deletion mutant in the mouse, a
targeting vector was designed to replace a 1167 bp DNA fragment that includes
the entire MFCS1 sequence in Lmbr1 intron 5 by a Neo cassette
(Fig. 2,
Fig. 3A). We obtained two lines
of germline chimeras from two targeted ES clones
(Fig. 3B), and obtained
MFCS1-deleted KO homozygotes by intercrossing the progeny carrying the KO
allele (Fig. 3C). Genotyping
revealed that KO homozygotes segregated in a Mendelian fashion (data not
shown). Although homozygous KO embryos are approximately the same size as
their wild-type littermates at E12.5, they display distally truncated,
vestigial limbs (Fig. 4A,B). By
E14.5, mutant embryos form thin, stick-like appendages that are
indistinguishable from those of Shh KO embryos
(Fig. 4D)
(Chiang et al., 1996;
Chiang et al., 2001
). However,
in contrast to the Shh KO mutants, which die at around E18.5 with
severe central nervous system defects, the MFCS1 KO homozygotes are viable,
and survive at least three months after birth
(Fig. 4F). Although the body
size of the mutant mice is slightly smaller than wild-type mice, they appear
healthy and can move freely using their shortened appendages, suggesting that
the deleted MFCS1 has a limb-specific function. The hindlimbs have one hard
digit with a nail on the dorsal side and a pad-like structure on the ventral
side (Fig. 4H,J), indicating
that the dorsoventral axis is properly established.
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Discussion |
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It is notable that the point mutations responsible for preaxial polydactyly
in humans and mice are scattered throughout the entire MFCS1 motif
(Lettice et al., 2003;
Sagai et al., 2004
). In the
two mouse mutants, Hx and M100081, ectopic Shh
expression is observed in anterior limb bud mesenchyme
(Masuya et al., 1995
;
Sagai et al., 2004
). This
suggests that there are multiple consensus motifs controlling Shh
expression, some of which are involved in the repression of Shh
expression in the anterior mesenchyme of limb buds, and others that are
responsible for its activation in the posterior mesenchyme of limb buds.
Mutations in the former probably lead to ectopic Shh expression in
the anterior margin of the limb buds, and preaxial polydactyly with
mirror-image digit duplication, whereas deletion of the entire conserved
sequence results in complete loss of Shh expression, as shown in the
present study. It is unclear why so many motifs are required for repressing
Shh expression in the anterior mesenchyme of developing limb buds. As
yet, no known consensus binding sequences have been identified in the MFCS1.
Moreover, no transcription factors are known to bind to the MFCS1.
Characterization of additional point mutations in the conserved sequence will
be necessary to elucidate the molecular mechanism responsible for long-range
cis-acting regulation.
In this study, we found that two other sequence blocks, MFCS2 and MFCS3, are highly conserved between medaka and mammals. The ordering of the three blocks relative to the Shh coding sequence is also conserved among the three species. It is possible that all three MFCS blocks cooperatively function in the cis-regulation of Shh expression in fins or limbs, although we cannot exclude the possibility that MFCS2 and MFCS3 are both involved in regulating Lmbr1 expression, or some other function altogether. Since there are no sequence motifs commonly shared among the three MFCS blocks, different trans-factors may bind to each MFCS block. At present, there is no evidence that human and mouse preaxial polydacytyly is caused by mutations in the MFCS2 and MFCS3 blocks. It would be of interest to examine the limb phenotype when these two sequence blocks are deleted in mice.
A notable feature of MFCS1 is its long distance from the coding sequence of
Shh. Another long-range cis-regulatory element, the local control
region (LCR) of the ß-globin locus, has been identified and well
characterized (Li et al.,
1999). A recent report showed that the LCR is in close physical
proximity to the ß-globin gene during active transcription, suggesting a
long-range interaction of the cis-regulatory element and the transcription
unit (Harrow et al., 2004
). It
has been reported that a LacZ reporter transgene, in which MFCS1 was
linked to a minimal ß-globin promoter, is sufficient to initiate
limb-specific expression at the proper time
(Lettice et al., 2003
).
However, the transgenic construct failed to terminate gene expression at the
appropriate time, suggesting that while the physical distance between the
cis-regulatory element and the Shh coding sequence is not essential
for the initiation of limb-specific Shh expression, proper
termination of transcription may require some distance between the two
elements (Lettice et al.,
2003
). At present, however, it is still unknown whether direct
communication is required between the long-range cis-regulatory element and
the Shh coding sequence.
MFCS1 KO mouse is a model for human acheiropodia
Severe truncation of distal skeletal elements of human limbs, resembling
the phenotype of the present MFCS1 KO mouse and the Shh KO mouse, is
known as acheiropodia, which maps to chromosome 7q36
(Ianakiev et al., 2001).
Acheiropodia is caused by a deletion of a 4-6 kb fragment including exon 4 of
human LMBR1 and its flanking region
(Ianakiev et al., 2001
). We
searched sequences conserved between human and mouse genomes in the
corresponding region, but failed to identify conserved non-coding regions with
sequence homology comparable with the three blocks of MFCS. Since the overall
phenotype of acheiropodia resembles that of MFCS1 KO homozygotes, it is
possible that in the human genome there is a human-specific cis-regulatory
element controlling limb-specific Shh expression in the vicinity of
exon 4 of LMBR1. Considering the similarity of the map position and
the phenotype, the MFCS1 KO mutant mouse is probably a useful animal model for
studying acheiropodia.
Limbless species have lost MFCS1
We have demonstrated that MFCS1 in the mouse contains a major limb-specific
enhancer of Shh that is essential for proper limb development. MFCS1
is also present in reptiles and amphibians as well as the teleost fish Medaka
(Sagai et al., 2004). A key
role of the cis-regulator for fin- and limb-specific Shh
expression may have placed constraints on MFCS1 to remain unchanged during the
evolution of the tetrapod and teleost lineages. Interestingly, we have
previously found that this sequence is lost in certain limbless species of
reptiles and amphibians, such as snakes and a limbless newt
(Sagai et al., 2004
). Given
our present data showing the importance of this enhancer in limb development,
it is possible that loss of the conserved intronic sequence MFCS1 represents
one way by which limblessness may have evolved in vertebrate species.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/4/797/DC1
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