To address the hypothesis that DUSP6 functions in vivo as a negative feedback regulator of MAPK signaling, we used gene targeting to disrupt mouse Dusp6. Homozygous mutant embryos showed variably penetrant increased levels of dpERK, the proposed DUSP6 substrate, in the limb bud, globally increased levels of Erm, a transcriptional target of the ERK pathway, as well as increased transcripts initiated from the Dusp6 promoter itself. Taken together, these molecular data are consistent with a pathway in which signals that activate ERK lead to increased transcription of Dusp6. The resulting DUSP6 protein then feeds back to dampen the signal by inactivating ERK.
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, 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 in 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 retroviral-mediated increases in Fgf3
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 in 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
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 suggests 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 the 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 discernable 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 (Dickinson et al., 2002b
, and data not shown). 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
) 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 SPROUTY, SEF, or SPRED, 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.