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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02701
1 Department of Human Genetics, University of Utah, 15 N 2030 E RM 2100, Salt
Lake City, UT 84112-5330, USA.
2 Division of Radiobiology, University of Utah, 729 Arapeen Drive #2334, Salt
Lake City, UT 84108-1218, USA.
Author for correspondence (e-mail:
suzi.mansour{at}genetics.utah.edu)
Accepted 17 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mkp3, Pyst1, Dual specificity phosphatase, Craniosynostosis, Middle ear, Otic capsule, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Thisse and Thisse,
2005). The responses to FGF signals are dosedependent. Just as a
reduction in FGF signals can lead to developmental abnormalities, so too does
an increase in FGF signaling. Indeed, some of the most frequently observed
human mutations are activating mutations in Fgfr genes that cause a
variety of dominant skeletal disorders
(Cohen, 2004;
Wilkie, 2005).
Many FGF signals travel and are amplified through the
extracellular-signal-regulated kinase (ERK) mitogen-activated protein kinase
(MAPK) pathway. FGF binding stimulates autophosphorylation of the FGFR, which
recruits signal adapters and leads to sequential phosphorylation and
activation of cytoplasmic protein kinases, ultimately resulting in the
diphosphorylation of ERK on threonine and tyrosine residues. This activated
form of ERK has substrates in all cellular compartments, including the
nucleus, where it phosphorylates and activates transcription factors,
effecting changes in gene expression
(Powers et al., 2000;
Chen et al., 2001;
Tsang and Dawid, 2004;
Eswarakumar et al., 2005).
Significantly, Corson and colleagues
(Corson et al., 2003) showed
that most of the diphosphorylated ERK (dpERK, an indicator of ERK pathway
activity) in early mouse embryos [embryonic day (E) 6.5-10.5] is dependent on
FGFR activity. This suggests that FGFs, as opposed to other signals that also
act through receptor tyrosine kinases, are the major input into the ERK
pathway at these stages.
Signaling through MAPK pathways can be attenuated at several levels and one
class of dual-specificity phosphatases, the MAPK phosphatases (MKPs) inhibit
MAPK signaling by dephosphorylating activated MAPKs. For example, in
Saccharomyces cerevisiae, mating pheromone-induced signaling through
the MAPK Fus3p induces the MKP Msg5p, which feeds back to turn off the signal
by inactivating Fus3p (Zhan et al.,
1997). Similarly, during Drosophila embryogenesis,
signals required for dorsal closure activate the DJNK (basket) MAPK pathway,
leading to transcriptional induction of the MKP puckered, which feeds back to
inactivate DJNK and dampen the signal
(Martin-Blanco et al., 1998).
In addition, Gomez and colleagues
(Gómez et al., 2005)
found that Drosophila MKP3 functions as a negative feedback regulator
of epidermal growth factor receptor-stimulated ERK signaling during wing vein
development.
Mammalian genomes contain at least 11 DUSP genes encoding the MKPs
(Alonso et al., 2004), several
of which have been analyzed biochemically. Some MKPs are relatively
non-specific toward different dpMAPKs in vitro. Other MKPs, however, show
substrate specificity, including the structurally related cytosolic MKPs -3
(DUSP6), -X (DUSP7) and -4 (DUSP9), which inactivate dpERK in preference to
other activated MAPKs (Camps et al.,
2000; Keyse, 2000;
Theodosiou and Ashworth,
2002). Dusp6 (also known as Mkp3 or
Pyst1) transcripts can, in some circumstances, be induced in cultured
cells by growth factors that stimulate the ERK pathway
(Mourey et al., 1996;
Muda et al., 1996a), but clues
to physiologic inducers have come from embryonic expression analyses. We and
others noticed that Dusp6/Mkp3 is expressed during embryonic
development of several vertebrate species in a pattern that corresponds with
areas of active FGF signaling, suggesting that it could be a conserved
transcriptional target of FGF signals
(Dickinson et al., 2002a;
Klock and Herrmann, 2002;
Eblaghie et al., 2003;
Kawakami et al., 2003;
Tsang et al., 2004;
Gómez et al., 2005)
(C.L. and S.L.M., unpublished). Indeed, ectopic FGF signals activate
Dusp6/Mkp3 transcription in chick, zebrafish and frog embryos, as
well as in explanted mouse neural tube cultures; ectopic Dusp6/Mkp3
expression reduces dpERK levels, and local siRNA, global morpholino-mediated
knockdown, or dominant-negative experiments suggest roles for
Dusp6/Mkp3 in chick limb development, axial patterning of zebrafish
embryos and anterior development of frog embryos, respectively
(Eblaghie et al., 2003;
Kawakami et al., 2003;
Tsang et al., 2004;
Echevarria et al., 2005;
Gómez et al., 2005;
Smith et al., 2005). Taken
together, these results show that Dusp6/Mkp3 can be activated by FGF
signaling and suggest a negative feedback role for DUSP6/MKP3 in FGF/ERK
signaling, but genetic loss-of-function data are lacking.
To address the hypothesis that mouse DUSP6/MKP3 plays a role in FGF-stimulated ERK signaling analogous to the MKPs that play clearly established negative feedback roles in regulating invertebrate MAPK signaling pathways, we studied Dusp6/Mkp3 expression in FGFR-deficient mouse embryos and generated and analyzed a targeted loss-of-function Dusp6/Mkp3 allele. Our data show that in mouse embryos, Dusp6/Mkp3 transcription depends on FGF signaling and that dpERK is a physiologic DUSP6/MKP3 substrate. Furthermore, we find that loss of Dusp6/Mkp3 leads to dominant, incompletely penetrant and variably expressive phenotypes that have features, including skeletal dwarfism, coronal craniosynostosis and hearing loss, in common with dominant gain-of-function mutations in human and mouse FGFRs.
Dusp6 is the official symbol for the mouse gene encoding MKP3 (MGI:1914853), and this nomenclature is used from here on.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Stained embryos were cryoprotected and sectioned at 14
µm as described (Wright and Mansour,
2003). The Dusp6 probe was complementary to nucleotides
1-413 of the Dusp6 reference sequence (5' UTR; GenBank
Accession number NM_02628). The Erm probe was complementary to
nucleotides 1309-1766 of the Etv5 reference sequence (GenBank
Accession number NM_023794). The Fgfr1 probe was complementary to
nucleotides 1-459 of the Fgfr1 cDNA (5' UTR; GenBank Accession
number BC010200). The Fgf8, Fgf10, Fgfr2b and Fgfr2c
isoform-specific and Fgfr4 probes were generated as described
(Wright et al., 2003;
Wright and Mansour, 2003;
Ladher et al., 2005). Whole
mouse embryos were stained with antibodies directed against diphosphorylated
ERK1/2 exactly as described by Corson et al.
(Corson et al., 2003).
Dusp6 gene targeting
A Dusp6-containing lambda genomic phage was isolated from a strain
129SV/J library (Stratagene). Standard techniques were used to isolate a 6.9
kb fragment containing the entire gene and transform it into a LacZ
knock-in gene targeting vector (see Fig.
3A). An nlsLacZSV40pA cassette was excised from a gene-trap vector
(Yang et al., 1997), excluding
the splice acceptor sequence, and inserted into the Dusp6 MscI site
in exon 1, fusing the first 56 codons of the DUSP6 open reading frame (ORF)
in-frame with the ßgal ORF. A self-excising Neor
expression cassette (ACN) (Bunting et al.,
1999) was placed immediately downstream of the LacZ gene
for positive selection of targeted cell lines. In anticipation of the small
possibility of spurious transcription initiation from inside the LacZ
cassette, followed by translation initiation from an internal Dusp6
AUG and/or the remote possibility of translation initiating downstream of the
DUSP6/ßGAL ORF on an SV40pA read-through transcript, we added a linker
containing a stop codon in the DUSP6 frame into the BstBI site in
exon 3. Any hypothetical DUSP6 fragment produced from such transcripts would
be missing portions of both the amino- and carboxy-terminal domains that are
essential for DUSP6 activity (Camps et al.,
1998; Zhou et al.,
2001). The targeting construct was flanked by two different
thymidine kinase expression cassettes for negative selection. The targeting
DNA was linearized and electroporated into R1-45 embryonic stem (ES) cells.
After selection in 380 µg/ml G-418 by weight (Invitrogen) and 2 mmol/l
ganciclovir (Sigma), drug-resistant colonies were cloned and expanded. DNA
isolated from each colony was screened by Southern blot hybridization using
NdeI digestion and a 3' flanking probe. DNAs showing the 7.0 kb
targeted band were further screened by PCR with primers 376
(5'-GGTATCAGCCGCTCTGTCAC-3') and 331
(5'-GGACACGGTTGTCACAAGG-3') for the presence of the stop codon in
exon 3. The wild-type allele was 187 bp and the mutant (stop-containing)
allele was 198 bp. DNAs from targeted cell lines that carried the stop codon
were further analyzed by Southern blotting using a variety of enzymes and
probes. Of 192 drug-resistant cell lines tested, 12 had a targeted insertion
in the Dusp6 locus and of these, eight also carried the stop
codon.
Dusp6 mutant mouse generation and genotyping
All work with mice complied with protocols approved by the University of
Utah Institutional Animal Care and Use Committee. Several correctly targeted
cell lines were aggregated with C57Bl/6 morulae, cultured overnight and
implanted into pseudopregnant females
(Khillan and Bao, 1997). Three
of these cell lines gave rise to chimeric males that transmitted the targeted
allele (less the self-excising Neo cassette) to offspring. In
subsequent crosses, the mutant and wild-type alleles were distinguished in
tail or yolk sac DNA using either of two 3-primer PCR mixes. The mix
containing primers 344 (5'-CTGTGTCGCTTTCCCTAACC-3'), 309
(5'-ACGCTGCTGCTGTTGC-3') and 315
(5'-GACCGACTCTCCACCAGTGT-3') produced a wild-type band of 499 bp
and a mutant band of 363 bp. The mix containing primers 344 (as above), 357
(5'-CCAGGGTTTTCCCAGTCA-3') and 430
(5'-CTAGCTCCCCTAAGCGCAAT-3') produced a wild-type band of 661 bp
and a mutant band of 522 bp. No difference between intercross offspring
produced from different targeted cell lines was immediately apparent; so one
line was selected for all further analysis and backcrossing.
Mice carrying the Fgfr1
(Fgfr1tm1Cxd,
MGI:2153353) and Fgfr2
IgIII
(Fgfr2tm1Cxd, MGI:2153790) hypomorphic alleles were
generously provided by Dr Chuxia Deng and genotyped as described
(Xu et al., 1998;
Xu et al., 1999).
Northern blot hybridization
Total RNA was isolated from E11.5 embryos or adult brains, mRNA was
purified and 3 µg of each genotype was analyzed by northern blot
hybridization according to standard protocols
(Yang et al., 2001). The 448
bp 3' UTR probe was generated by PCR amplification of mouse genomic DNA
with primers 311 (5'-ACCCCTTGAGACACTGTAAGC-3') and 329
(5'-GGGTATAGTGGAGCCAAAGAGA-3'). The 5' UTR probe was the
same as that used for in situ hybridization. The LacZ probe was
generated by PCR amplification of plasmid DNA with primers 24
(5'-GGGTTGTTACTCGCTCACA-3') and 25
(5'-AAAGCGAGTGGCAACATGG-3').
Skeleton preparation, computed tomography (CT) scanning and bone histology
To visualize the skeleton, animals were asphyxiated in CO2,
skinned, fixed in 95% ethanol, defatted in acetone and then stained with
Alcian Blue 8GX and Alizarin Red S and cleared as described
(Mansour et al., 1993).
MicroCT scans of pups sacrificed at P10 were performed as described
(Keller et al., 2004). Cranial
vault measurements were made using reconstructed mid-sagittal sections. For
bone histology, the proximal tibial metaphyses were stained en bloc with
Villanueva bone stain, dehydrated in graded concentrations of alcohol,
defatted in acetone and embedded in methyl methacrylate monomer. Longitudinal
frontal sections of the tibia were cut at 4 µm. One set of sections was
stained with 5% silver nitrate and counterstained with 0.5% basic fuchsin,
another set of sections was stained with 0.1% Toluidine Blue O
(Jee et al., 1997).
Auditory brainstem response threshold measurements
Mice were anesthetized using 0.02 ml/g Avertin. Auditory brainstem response
(ABR) thresholds for click (47 µs), 8 kHz, 16 kHz and 32 kHz tone pip
stimuli (3000 µs, Exact Blackman envelope) were determined according to
Zheng et al. (Zheng et al.,
1999), using high frequency transducers controlled and analyzed by
SmartEP software (Intelligent Hearing Systems).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Klock and Herrmann, 2002;
Eblaghie et al., 2003;
Kawakami et al., 2003;
Tsang et al., 2004). Areas of
the embryo potentially competent to respond to FGF signals can be recognized
by their expression of FGF receptor genes (Fgfrs). If Dusp6
is a transcriptional target of FGF signaling, then it should be coexpressed
with Fgfrs. We found that between E9.5 and 11.5, Dusp6
expression was highly correlated with several areas of Fgfr
expression (Fig. 1). For
example, Fgfr1 and Dusp6 were expressed in the branchial
arches and limb buds at E9.5 (Fig.
1A,B), Fgfr4 and Dusp6 were expressed in the
somites at E10.5 (Fig. 1D,E)
and Fgfr2b and Dusp6 were expressed in the branchial arches
and otic vesicle at E11.5 (Fig.
1G,H).
One of the intracellular pathways by which activated FGF receptors signal
to the nucleus, is the ERK pathway, activation of which can be visualized by
immunostaining for dpERK. Indeed, most expression domains of dpERK in
E7.5-10.5 mouse embryos depend on FGF signaling
(Corson et al., 2003). If
Dusp6 is a transcriptional target of FGF signaling through the ERK
pathway, then it should be coexpressed with dpERK. We found that there was a
striking correlation between the dpERK and Dusp6 expression domains
between E9.5 and 11.5 (Fig. 1).
For example, dpERK and Dusp6 were found in the limb buds, branchial
arches and midhindbrain boundary at E9.5
(Fig. 1B,C), in the somites,
limb buds and branchial arches at E10.5
(Fig. 1E,F) and in the limb
buds, branchial arches, otic vesicles and olfactory pits at E11.5
(Fig. 1H,I).
Sections taken through the developing limb buds (Fig. 1J-O) and branchial arches (Fig. 1P-S) at E10-10.5 illustrate potential FGF signaling pathways in more detail. In the limb, the genes encoding the ligands FGF8 (Fig. 1J) and FGF10 (Fig. 1M) were expressed in the apical ectodermal ridge (aer) and mesenchyme (mes), respectively. FGF8 and FGF10 could signal to their preferred receptors, mesenchymal FGFR2c (Fig. 1K) and ectodermal FGFR2b (Fig. 1N), respectively. Dusp6 and dpERK were primarily mesenchymal (Fig. 1L,O), suggesting that they may participate in the mesenchymal response to FGF signals. Similar FGF signaling pathways were present in the branchial arches. FGF8 and FGF10 were ectodermal and mesodermal, respectively (data not shown), whereas genes encoding their receptors, FGFR2c (Fig. 1Q) and FGFR2b (Fig. 1P), were expressed in the mesoderm and ectoderm, respectively. As in the limb bud, branchial arch Dusp6 and dpERK were primarily mesenchymal (Fig. 1R,S). Taken together, these data show that Dusp6 expression correlates with Fgfr expression domains and with sites of FGF-activated ERK signaling in the mesenchyme.
Dusp6 expression in mouse embryos depends on FGF signaling
Ectopic activation of FGF signaling in zebrafish and chick embryos or mouse
neural tube explants is sufficient to induce ectopic Dusp6 expression
(Eblaghie et al., 2003;
Kawakami et al., 2003;
Tsang et al., 2004;
Echevarria et al., 2005;
Smith et al., 2005).
Furthermore, genetic or physical ablation of FGF4 and FGF8 signals from the
aer in mouse, chick and zebrafish embryos
(Eblaghie et al., 2003;
Kawakami et al., 2003),
application of the FGF receptor inhibitor SU5402 to developing chick somites
or mouse neural tube explants (Smith et
al., 2005; Vieira and
Martinez, 2005), or expression of dominant-negative FGF receptors
in zebrafish embryos (Tsang et al.,
2004) reduces endogenous Dusp6 expression in the target
tissue. To determine whether FGF signaling is required globally for
Dusp6 transcription in mouse embryos, we assayed Dusp6
expression by in situ hybridization of whole mouse embryos homozygous for
hypomorphic mutations in either Fgfr1 or Fgfr2
(Xu et al., 1998;
Xu et al., 1999).
Dusp6 transcripts were severely reduced in both E9.5
Fgfr1 (Fig.
2A,B) and E8.5 Fgfr2IgIII
(Fig. 2C-H) homozygous embryos
relative to similarly staged control embryos stained under identical
conditions. These data show that Dusp6 expression requires signaling
through FGFR1 or FGFR2.
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). To determine the role of mouse Dusp6, its function
was disrupted using standard gene targeting techniques to insert an inframe
LacZ cassette into the first coding exon and a stop codon in the
third exon (Fig. 3A). Correctly
targeted ES cell lines were identified by Southern blot hybridization analysis
of NdeI-digested DNA using 3' flanking and internal
Cre probes (Fig. 3B).
Additional digestions and probes confirmed the expected structure of the
targeted allele (data not shown). Correctly targeted cell lines were further
screened for the presence of the stop codon by PCR using primers flanking the
newly introduced stop codon (Fig.
3C). During germline transmission of the correctly targeted, stop
codoncontaining allele, the Neo gene was automatically deleted and
this was confirmed by Southern hybridization analysis (data not shown). The
LacZ knock-in allele was designated Dusp6L. A
3-primer PCR assay was developed to distinguish the wild type (+) and mutant
(-) alleles (Fig. 3D).
To determine the efficacy of the disruption strategy, we hybridized a
northern blot of E11.5 mRNAs with a Dusp6 exon 3 3' UTR probe
(Fig. 3E). The expected 
3
kb Dusp6 transcript and an uncharacterized, weakly expressed 4
kb transcript were evident in wild-type and heterozygous samples, but were
absent from homozygous mutant mRNA, showing that the targeting strategy
effectively disrupted production of Dusp6 mRNA. A novel, lowabundance
transcript of 7.5 kb was apparent in heterozygous and mutant samples.
This is the expected size for mutant transcripts initiated at the
Dusp6 promoter and terminated at the Dusp6, rather than the
SV40 polyadenylation signal, followed by removal of the Dusp6 introns
(see Fig. 4G,I for further
hybridization data supporting this interpretation). Even if translation of
this 7.5 kb fusion mRNA could initiate at a Dusp6 AUG codon
downstream of the DUSP6/ßgal ORF, a functional DUSP6 protein could not be
produced from the mutant allele, as portions of both the amino- and
carboxy-terminal regulatory domains required for DUSP6 activity would be
absent (Camps et al., 1998;
Zhou et al., 2001). Thus, the
targeted allele is likely to be null.
One objective of the LacZ knock-in strategy was to enable a simple reporter assay for Dusp6 expression. Surprisingly, we found that heterozygous embryos exhibited little or no ßgal activity, whereas homozygous E10.5-16.5 embryos produced readily detectable ßgal in a pattern that largely, though not perfectly, mimicked that of transcripts detected by whole-mount in situ hybridization in embryos of both genotypes by using either Dusp6 5' UTR or LacZ probes (data not shown, but see Fig. 4G for northern data). This apparently post-transcriptional phenomenon is under investigation.
Dusp6 is not required for embryonic development, but has incompletely penetrant effects on dpERK, Erm and Dusp6 levels
To assess the role of Dusp6 in embryonic development, the F1
Dusp6+/L offspring of germline chimeras were intercrossed
and embryos were collected and genotyped between E8.5 and 17.5. Of 1160
intercross offspring, 244 were wild type, 459 were heterozygous, 226 were
homozygous mutant and 11 genotypes could not be determined. These results are
consistent with a normal Mendelian distribution of wild-type and mutant
alleles. No obvious abnormalities were seen in embryos of any genotype,
indicating that Dusp6 does not have a unique and visibly evident
function during embryonic mouse development.
DUSP6 has a high degree of substrate specificity in vitro,
dephosphorylating dpERK in preference to other MAP kinases
(Groom et al., 1996;
Muda et al., 1996b). To
determine whether ERK signaling was perturbed in homozygous mutants, we
stained whole embryos at E9.5 and 10.5 with antibodies directed against dpERK
in parallel with wild-type littermate control embryos. In four out of eight
homozygotes analyzed, the extent of dpERK staining was increased, particularly
in the limb (Fig. 4A-C). In the
remaining cases, no differences in dpERK staining between wild-type and
homozygous mutant embryos could be discerned (data not shown). Thus, as
predicted, DUSP6 dephosphorylates dpERK in vivo.
One of the responses to activation of the ERK pathway is an increase in
Erm (Etv5 - Mouse Genome Informatics) transcripts
(Raible and Brand, 2001;
Roehl and Nüsslein-Volhard,
2001; Firnberg and
Neubüser, 2002; Liu et
al., 2003). To determine whether Erm expression was
affected in Dusp6 mutants, we subjected E10.5 embryos (nine of each
genotype) to RNA in situ hybridization analysis with an Erm probe.
After an identical staining reaction, stopped when Erm transcripts
were just becoming evident in wild-type embryos
(Fig. 4D), three out of nine
heterozygous embryos had greater Erm expression
(Fig. 4E) and three out of nine
homozygotes had even more Erm signal
(Fig. 4F). No differences from
wild-type controls were apparent in the remaining heterozygous or homozygous
embryos. These results confirm that inactivation of Dusp6 can
increase signaling through the ERK pathway. Furthermore, the intermediate
phenotype seen in heterozygotes suggests that signaling through the ERK
pathway to induce Erm is sensitive to the level of
Dusp6.
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5 kb and 7.5 kb, reflecting
production of Dusp6/LacZ fusion transcripts with polyadenylation
probably occurring at the SV40 or Dusp6 polyadenylation sites,
respectively (Fig. 4G-I). This
result shows that Dusp6 transcription is subject to negative
autoregulation.
Loss of Dusp6 results in dominant, incompletely penetrant postnatal lethality
To determine whether Dusp6 is required for any aspect of postnatal
development, Dusp6+/L offspring of chimeric males and
C57Bl/6 females were produced and offspring of these F1
Dusp6+/L intercrosses were genotyped at weaning (4
weeks postnatal). The numbers of wild-type and heterozygous F1 progeny of the
chimeras did not deviate significantly from those expected
(Table 1). By contrast, of 580
F1 intercross offspring genotyped, 168 were wild type, 287 were heterozygotes
and only 125 were homozygotes (Table
1), all of which appeared normal. This genotypic distribution
deviated slightly, but significantly, from the Mendelian expectation
(P=0.04) and suggested reduced viability of homozygotes and possibly
even heterozygotes. Increasing the C57Bl/6 contribution to the genetic
background by crossing F1 heterozygotes to C57Bl/6 animals caused a
significant reduction in the observed numbers of heterozygous offspring
relative to wild type. Of 540 F1 x C57Bl/6 offspring, 309 were wild type
and only 231 were heterozygous (P=0.00078,
Table 1). The genotypic
distribution of the F2 Dusp6+/L intercross offspring was
even more significantly distorted than that of the F1 intercross. Among 505
total offspring, 142 were wild type, 269 were heterozygous and 94 were
homozygous mutant (P=0.0036, Table
1). The very mixed genetic background of this intercross may
explain why the heterozygous lethality seen in the backcross was not obvious.
Taken together, these breeding results suggest that the Dusp6
mutation is associated with dominant, but incompletely penetrant, postnatal
lethality that is exacerbated on the C57Bl/6 background.
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As small Dusp6 mutants did not appear to have any physical
impediments to feeding, such as malocclusion or cleft palate (data not shown),
we examined their long bones for disturbances of the growth plate. The
proximal tibia of a P14 wild-type animal had typical histology, with
well-ordered chondrocytes proceeding through proliferative, hypertrophic and
ossification stages (Fig.
5B,D). By contrast, the proximal tibia of a small P14
Dusp6-/- pup had severely reduced hypertrophic and
ossification zones (Fig. 5C).
In addition, chondrocytes in the mutant proliferating zone were disorganized;
they did not form typical long straight columns of cells
(Fig. 5E). This phenotype was
observed in all three small mutants examined and is very similar to that of
mice bearing activating mutations in FGFR3
(Brodie and Deng, 2003).
Loss of Dusp6 causes craniosynostosis
Mildly activating mutations in FGFR1 and FGFR2 typically cause
craniosynostoses (premature fusions of the cranial sutures) in humans and
mice, and hand and foot malformations in humans. In addition, the P250R
substitution in FGFR3 causes `non-syndromic' craniosynostosis in humans
(Zhou et al., 2000;
Brodie and Deng, 2003;
Chen et al., 2003;
Wang et al., 2005;
Wilkie, 2005). Therefore, we
examined small Dusp6+/L and Dusp6L/L
animals for additional phenotypes associated with activation of FGF signaling.
Staining the carcasses of small P5-15 animals to reveal cartilage and bone
showed coronal craniosynostosis in small heterozygous and homozygous
individuals (Fig. 5G,I), but
not in wild-type (Fig. 5F,H) or
normally sized Dusp6+/L or Dusp6L/L
pups (data not shown). Furthermore, microCT scanning of small P10
Dusp6+/L and Dusp6L/L skeletons showed
that the skull vault height:length and height:width ratios were significantly
larger than those of the corresponding wild-type control ratios, which is
consistent with mild craniosynostosis
(Table 2).
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Loss of Dusp6 causes hearing loss
Clinical descriptions of patients with dominant, activating FGFR mutations
often include reports of sensorineural and/or conductive deafness
(Gorlin, 2004). Therefore, we
measured ABR thresholds at about 6 weeks of age in normally sized animals of
all three Dusp6 genotypes, but no significant differences between
genotypes were observed (data not shown). In addition, five small intercross
offspring survived to 21-28 days and could be evaluated for an ABR. Four of
the five small animals had ABR thresholds that were significantly elevated in
one or both ears relative to wild-type littermates and other similarly aged
control mice (Table 3).
Examination of inner ear histology from a small heterozygote (I607) with
bilaterally increased ABR thresholds did not reveal any obvious abnormalities
relative to a normal hearing control littermate, whereas in another small
bilaterally affected heterozygote (I1034), the opening of the middle ear
cavity, to which the tympanic membrane attaches, was distorted
(Fig. 6A,B). That animal had
ossicles that appeared normal (data not shown). Similar results were obtained
for the left (affected) ear of the unilaterally affected heterozygote, I1008.
Another unilaterally affected homozygote (I919) died unattended before any
tissues could be recovered for analysis.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One signal that initiates the negative feedback loop is likely to be FGF,
because we found that Dusp6 transcription depends on signaling
through FGFR1 or FGFR2. Furthermore, we found that abrogation of
Dusp6 function in mice leads to variably penetrant and expressive
postnatal phenotypes that are similar to some of the features of ectopic FGF
ligand expression or dominant activating mutations in FGFRs, which are major
inputs into the ERK pathway (Powers et
al., 2000; Tsang and Dawid,
2004; Eswarakumar et al.,
2005). Beginning around P5, affected Dusp6 mutants were
small, exhibited coronal craniosynostosis and middle ear and otic capsule
malformations, and affected individuals that survived past P21 frequently had
uni- or bilateral hearing loss.
Skeletal dwarfism manifesting in the early postnatal period is also
characteristic to various extents of mice carrying Fgf2- or
Fgf9-expressing transgenes (reviewed by
Ornitz and Marie, 2002), and
of knock-in mice carrying the Apert syndrome equivalent mutation (FGFR2S252W)
(Chen et al., 2003;
Wang et al., 2005), and all of
the characterized FGFR3 syndromic gain-of-function mutations
(Brodie and Deng, 2003). In
addition, osteoglophonic dysplasia, which can be caused by any of several
activating mutations in FGFR1
(White et al., 2005), is
characterized by short stature. In general, the more strongly activating
receptor mutations lead to the strongest dwarfing phenotypes
(Ornitz and Marie, 2002;
Chen and Deng, 2005). This is
consistent with the idea that FGF signaling limits endochondral bone growth
and that inactivating mutations in negative regulators of FGF signaling, such
as Spred2 (Bundschu et al.,
2005) or Dusp6, lead to increases in FGF signaling and
decreased bone growth. The bone histology of small Dusp6 mutants
(reduced hypertrophic and ossification zones, disorganized proliferation zone)
is more similar to that of mice with Fgfr3 gain-of-function mutations
(e.g. Li et al., 1999) than to
the Apert knock-in models, which in one case showed a slightly reduced
proliferation zone (Chen et al.,
2003) and in the other subtle irregularity of the hypertrophic
zone (Wang et al., 2005). This
suggests that DUSP6 is more likely to regulate signaling downstream of FGFR3
than of FGFR2 in the growth plate. It would be interesting to make similar
comparisons of growth plate histology when a mouse model of osteoglophonic
dysplasia is produced.
The craniosynostosis seen in Dusp6 mutants is also likely to be
FGF-mediated, as similar phenotypes are seen consequent to ectopic
Fgf2 expression or retrovirally mediated increases in Fgf3
and Fgf4 expression. In addition, many of the models of FGF receptor
activation have more severe craniosynostoses, in some instances involving the
coronal as well as the interfrontal and sagittal sutures (reviewed by
Ornitz and Marie, 2002;
Chen and Deng, 2005), than do
affected Dusp6 mutants, in which only the coronal suture is affected.
Suture formation is regulated by opposing FGF signaling pathways that control
the balance of cellular proliferation (through FGFR2) and differentiation
(through FGFR1) (Iseki et al.,
1999). Thus, it is conceivable that Dusp6 has
differential effects on the two pathways, perhaps regulating signaling through
FGFR1 rather than through FGFR2 in the developing calvarium.
No information on middle ear or otic capsule morphology or auditory status
is available for any of the mouse Fgf or Fgfr
gain-of-function mutations, but the common findings of hearing loss and
otopathology in Apert, Pfeiffer, Crouzon and Muenke syndrome patients
(Gorlin, 2004) and the mouse
Dusp6 mutant phenotype suggest that these pathologies may yet be
found in the mouse models as well. Apert and Pfeiffer syndromes are
characterized by limb malformations (syndactyly and broad first digits,
respectively) (Muenke and Wilkie,
2000; Wilkie,
2005), but no such malformations were apparent in the small
Dusp6 mutants. This may not be surprising, as none of the mouse
models for these FGFR1 and FGFR2 syndromes have limb findings, potentially
reflecting slight differences in the regulation of FGF signaling between mice
and humans.
Taken together, the Dusp6 mutant phenotypes we observed do not precisely mimic any particular mouse Fgfr gain-of-function mutation, suggesting the possibility that Dusp6 is downstream of more than one FGFR, but that it does not serve as a negative feedback regulator of all FGF signaling events. Our finding that hypomorphic mutations in either Fgfr1 or Fgfr2 reduce, but do not entirely eliminate, Dusp6 expression at early embryonic stages further supports this idea. Genetic interaction studies between the Dusp6 mutant allele and various Fgf or Fgfr mutations could be used to address this hypothesis and learn which specific FGF signaling events are subject to regulation by DUSP6. Indeed, our preliminary studies suggest that loss of Dusp6 exacerbates the small size and lethality of the Fgfr1P250R allele. Whether this effect is a result of changes to craniofacial or limb skeletal elements or both is currently under investigation.
The variable penetrance of the embryonic molecular and postnatal
morphologic phenotypes resulting from Dusp6 loss, and the absence of
discernible morphologic changes in the mutant embryos, suggest that there
could be redundant ERK phosphatases that compensate to some extent for
Dusp6. These may or may not be part of a negative feedback loop
regulating ERK. DUSP7 and DUSP9 are both relatively ERK-specific in vitro
(Dowd et al., 1998;
Dickinson et al., 2002b), and
their transcripts have some areas of overlap with Dusp6, particularly
in the developing limb buds and branchial arches (data not shown)
(Dickinson et al., 2002b). The
Dusp7 mutant phenotype has not yet been reported, but mutation of the
X-linked Dusp9 gene leads to failure of placental development and
consequent lethality. Tetraploid rescued embryos develop normally, however
(Christie et al., 2005). Thus,
conditional Dusp9 (and Dusp7) alleles will have to be
generated in order to assess potential redundancy with Dusp6.
Finally, the similarity between the Dusp6 mutant phenotypes and those of humans with activating FGFR mutations suggests that mutations in DUSP6 or other negative regulators of FGF signaling, such as SPRY genes, SPRED genes or SEF (IL17RD - Human Gene Nomenclature Database), or indeed other DUSP genes, are good candidates for molecularly unexplained cases of FGFR-like syndromes. Furthermore, the increasing lethality of the Dusp6 mutation as the allele was backcrossed to C57Bl/6 shows that there are likely to be genes that interact with Dusp6 and suggests that genetic variation among negative regulators of FGF signaling is a potential source of modifiers of the variable expressivity of human FGFR mutations.
|
ACKNOWLEDGMENTS |
|---|
and Fgfr2IgIII alleles,
Charles Keller for carrying out the microCT scans, Webster Jee for assistance
with the bone histology and Lisa Urness for critiquing the manuscript. Xiaofen
Wang provided excellent technical assistance with mouse production; Albert
Noyes generously provided the dpERK data for
Fig. 1C; and Samantha Covington
generated the plasmid bearing the Fgfr1 5' UTR fragment. This
work was supported by grants from the NIH/NIDCD: R01DC02043 and R01DC04185,
from the Deafness Research Foundation, and from the Children's Health Research
Center. D.A.S. was a Primary Children's Medical Foundation Scholar. |
Footnotes |
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