haploinsufficient mice and other disease models, quantitative trait loci (QTL) analysis has been a successful approach to map a number of loci that modify the severity of disease traits, however, identification and confirmation of the causative sequence variation is labor intensive and has only been completed for a small number of mapped QTLs (28
). Additionally, recent data suggest that variation among the classical inbred strains is limited and not evenly distributed throughout the genome (29
), thus leaving large regions of the genome without the sequence diversity needed to be screened using current QTL methodologies. One way to increase the repertoire of genetic variation that can be tested for modifier effects is to introduce genome wide mutations into genetic crosses where the phenotype of interest has been well characterized, thus allowing potential modifier effects for every locus in the genome to be screened. Historically, this type of mutagenesis screen to identify enhancers and suppressors of phenotypes has been extremely successful in lower model organisms (30
) and more recently applied in mice to identify factors involved in selective biological processes (31
). Indeed, these mutagenesis screens have some distinct advantages over QTL analysis for the identification of specific sequence variations that modify a phenotype of interest (33
) including the relative ease with which the causative sequence variation can be identified.
In this paper, we present a sensitized mutagenesis screen to identify candidate genes for human neurocristopathies. Using this dominant screen we identified three loci that increased the severity of neurocristopathy in Sox10 haploinsufficient mice and have mapped these loci to regions not previously attributed to known WS loci. By including Sox10 haploinsufficiency in the screen, we can identify mutations with no heterozygote phenotype on their own, which otherwise could only be detected with an additional generation of breeding (~10 weeks in mice) that is required for a traditional three-generation recessive screen. In addition, our screen retains mutations that could be missed in a three-generation recessive screen due to embryonic lethality, which was in fact observed for all 3 of the loci reported here. The use of two different inbred strains in the screen eliminated the outcrossing required to map loci, thus allowing us to quickly determine if our phenotypes are caused by mutations in novel loci or represent mutations in previously identified NC development and disease genes.
One of the pedigrees identified in our ENU screen, Mos1, showed semidominant hypopigmentation and polydactyly and resulted in homozygous embryonic lethality. The extent of hypopigmentation was significantly increased in Mos1/+; Sox10LacZ/+ double heterozygotes compared with mice carrying heterozygous mutations in either single gene suggesting a synergistic interaction between the two loci. Subsequent linkage and candidate gene analysis identified a causative point mutation in Gli3. We showed that a previously published null allele of Gli3, Gli3Xt-J, had similar effects on pigmentation and survival. Additionally, a complementation test done by intercrossing Gli3Xt-J/+ and Gli3Mos1/+ mice failed to produce any viable compound heterozygous mice carrying both mutations, thus providing strong evidence that the stop mutation identified in Gli3Mos1 results in a functionally null allele that is responsible for the hypopigmentation in the Mos1 mice. Double heterozygous mice carrying mutations in both Sox10 and Gli3 show a significant increase in hypopigmentation compared with the Sox10LacZ/+ heterozygous mice. In particular, double heterozygous Sox10LacZ/+;Gli3Mos1/+ mice exhibit extensive ventral hypopigmentation that can extend over the dorsal surface to form a belt. Using Mitf, Si, DctLacZ and Sox10LacZ expression analysis, we showed that Gli3 deficiency results in a vast reduction of melanoblasts in the trunk.
is a member of the GLI family of C2H2-type zinc finger transcription factors whose members are vertebrate homologs of the Drosophila
Cubitus interruptus (Ci) gene (34
). GLI family members mediate the final stages of HH signaling and their regulation of HH target genes plays an important role during embryogenesis (reviewed in 35
). The timing and location of Gli3
expression in the dorsal neural tube overlaps with where NC cells form (20
), consistent with a role for GLI3 in specification of NC derivatives. However, the reduction in melanoblast specification in Gli3Mos1/Mos1
embryos is unlikely to result from an overall reduction of NC, since DRG and sympathetic ganglia appear relatively normal in these regions. Our observation that GLI3 deficiency does not impair later stages of melanocyte differentiation is consistent with an early role for GLI3 during specification of the melanocyte lineage. Interestingly, melanoblast specification outside the trunk region of Gli3Mos1/Mos1
embryos was not noticeably reduced. This embryonic phenotype is consistent with the phenotype in Sox10LacZ/+; Gli3Mos1/+
adult animals where hypopigmentation is limited to the trunk. This region-specific effect on melanoblast specification could be reflective of normal, wild-type melanoblast distribution, where lower melanoblast numbers are seen in the mid-trunk region (21
). Alternatively, there could be independent pathways that compensate for the loss of GLI3 in regions outside the trunk. Collectively, our data provides strong evidence supporting a role for Gli3
in early specification of the melanocyte lineage.
During development, GLI3 can act as either a transcriptional activator or repressor, and HH signaling regulates this dual activator/repressor function. In the presence of HH, full-length GLI3 activates target genes, while in the absence of HH, posttranslational cleavage of the C-terminus of GLI3 produces an N-terminal form of GLI3 that acts as a repressor (26
) (reviewed in 38
). Given that the NC is induced at the dorsal neural tube in a region with low HH activity, we predict that the GLI3 repressor would be the main GLI3 protein product involved in melanoblast specification. In support of this hypothesis, Gli3D699/D699
mutant embryos, which specifically express only the C-terminally truncated GLI3 repressor (27
), had normal numbers of trunk melanoblasts. These results show that the repressor form of GLI3 promotes melanoblast specification, and suggest that a low level of HH signaling is required for normal melanoblast specification.
The precise mechanism through which the GLI3 repressor acts to facilitate melanoblast specification remains to be determined. Because Sox10
interacts with a number of genes during melanocyte development, including Mitf
) and components of the wingless-related MMTV integration site (Wnt) signaling pathway (44
), the genetic interaction we observe between Gli3
mutants could be mediated through these other pathways rather than a direct interaction with Sox10
. Recent work reveals that canonical Wnt signaling may directly activate Gli3
, thereby helping to establish dorsal ventral patterning within the spinal cord (46
). Interestingly, the GLI3 repressor has been shown to inhibit canonical Wnt signaling by physically interacting with and antagonizing active forms of β-catenin (47
). Thus, Gli3
appears to be both a target of and a regulator of canonical Wnt signaling. The careful balance of HH and Wnt signaling was shown to affect neuronal subtypes within the developing spinal cord (46
) suggesting that perhaps interactions between the HH and Wnt signaling cascades could also play a role in melanocyte progenitor cell fate. Wnt signaling is known to influence the proliferation and specification of melanocyte precursors (48
), and it is possible that loss of GLI3 repressor in Gli3Mos1/Mos1
mutants perturbs the balance of HH and Wnt signaling, resulting in a ventralization of cell fate that affects the melanocyte lineage.
As a well-established downstream target and mediator of Wnt signaling (53
provides an intriguing direct target for GLI3 repression within the melanocyte lineage. However, a preliminary search for GLI3 binding sites did not identify any highly conserved GLI3 binding sites within the Mitf
promoter suggesting that the GLI3 repressor acts indirectly to regulate Mitf
expression. While it is likely that GLI3 interacts with a number of target genes during NC lineage specification, Foxd3
is a promising candidate for a GLI3 target and we hypothesize that this binding could indirectly regulate Mitf
genes have been implicated in mediating HH signaling in craniofacial NC derivatives (56
) and the zebrafish foxd3
directly binds the mitfa
promoter, thereby repressing transcription and inhibiting mitfa
-positive melanoblast specification (57
). Therefore it is possible that in Gli3Mos1/Mos1
expression is deregulated, thus disrupting melanoblast specification. While significant future work remains to determine the mechanism through which the GLI3 repressor affects melanoblast specification, our findings clearly implicate GLI3 as a participant in these networks and uncover a new role for GLI3 in the melanocyte lineage.
In conclusion, we have identified three loci that act as modifiers for Sox10-dependent melanocyte defects, increasing both penetrance and severity of the defects in Sox10LacZ/+ heterozygous mice. We have shown that one of these loci is a mutation in Gli3 and have demonstrated the phenotype caused by a reduction in normal Gli3 gene dosage is significantly exacerbated by a reduction in Sox10 gene dosage (Sox10LacZ/+ mice), suggesting that the two genes cooperate, directly or indirectly, during specification of the melanocyte lineage. These data highlight the role of Gli3 signaling in melanocyte development, and predict the importance of future studies investigating the role of GLI3 and/or SOX10 in other human disorders that combine digit and melanocyte defects.